Anti-orthopoxvirus recombinant polyclonal antibody

ABSTRACT

Disclosed is an anti-orthopoxvirus recombinant polyclonal antibody comprising distinct members which in union are capable of binding at least three orthopoxvirus related antigens, a pharmaceutical composition comprising the antibody, and a method for its production. Also disclosed is a polyclonal cell line capable of producing the recombinant polyclonal antibody as therapeutic methods utilizing the polyclonal antibody. Finally, the invention also pertains to a method for screening for useful V H  and V L  pairs useful when preparing the polyclonal antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/633,070, filed Dec. 4, 2006, which claims the benefit of DanishApplication No. PA 2005 01720, filed Dec. 5, 2005, both of which areincorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing in ASCIItext file (Name: Sequence_listing_(—)2488.0020001.ascii.txt; Size:117,921 bytes; and Date of Creation: Dec. 10, 2010) filed with theapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a recombinant polyclonalanti-orthopoxvirus antibody (anti-orthopoxvirus rpAb), in particular arecombinant polyclonal anti-vaccinia virus antibody (anti-VV rpAb). Theinvention also relates to polyclonal expression cell lines producinganti-orthopoxvirus rpAb or anti-Vs/rpAb. Further, the applicationdescribes diagnostic and pharmacological compositions comprisinganti-orthopoxvirus rpAb or anti-VV rpAb and their use in prevention, andtreatment of adverse effects of vaccination, or diagnosis and treatmentof orthopoxvirus infections.

2. Background Art

Smallpox is caused by airway infection with the orthopoxvirus, variola.The threat of smallpox outbreaks as a result of bioterrorism and theemergence of related viruses such as monkeypox, have revived the needfor anti-orthopoxvirus therapeutics and vaccination. Vaccinia virusvaccination mediates moderate to severe adverse reactions inapproximately one in every 1000. These are currently treated withanti-vaccinia virus immunoglobulin (VIG) isolated from donors with ahigh antibody titer. However, the estimated incidence of adverse effectsresulting from a general vaccination program using live attenuatedvaccinia virus exceeds the current production capacity of VIG, therebypreventing vaccination as an approach for public protection againstsmallpox. Furthermore, VIG has a very low specific activity resulting ina need for injection of large volumes. There is also the risk oftransmission of viral diseases from serum derived VIG products, as wellas problems with batch-to-batch variations. Therefore, investigations ofpossible alternatives for providing protecting against vaccinia virusadverse effects or infections by other orthopoxviruses have beenconducted.

Orthopoxviruses produces two types infectious particles, namely theIntracellular Mature Virions (IMV) and the Extracellular EnvelopedVirions (EEV). IMV plays a predominant role in host-to-host transmissionand EEV plays a major role in virus propagation within the host. The IMVparticle is assembled in the cytoplasm of infected cells and consists ofa virally induced membrane surrounding the genome containing ahomogenous core particle. EEV particles are generated by wrapping of IMVparticles in a host cell-derived membrane followed by egress of the EEVparticle. At a later stage the vaccinia virus infection results in celldeath and release of the infectious IMV particles. Viral proteinspresented at the surface of IMV or EEV particles are potential targetsfor antibodies, a total of five IMV-specific proteins and twoEEV-specific proteins have been reported to elicit virus neutralizingand/or protective effects when used for immunization or vaccination.Additionally, neutralizing and protective effects have been observed forthe passive administration of antibodies which specifically bind theseproteins (summarized in Table 1).

TABLE 1 Antigen Virion Antibody effect Immunization/vaccination effectA27L IMV + neutralize^(11, 12, 13) DNA: + neutralize ÷ protective⁶ (P14)(+) protective¹¹ Protein: + neutralize + protective^(1, 8) −protective¹⁵ A17L IMV + neutralize¹³ (P21) L1R IMV +neutralize^(10, 14, 15) DNA: + neutralize (+) protective³ (P25-29) +protective^(10, 15) Protein: + neutralize + protective² D8L IMV +neutralize¹⁵ Protein: ÷ neutralize + protective¹ (P32) ÷ protective¹⁵H3L IMV + neutralize^(4, 9) (P35) A33R EEV ÷ neutralize^(10, 15) DNA: ÷neutralize + protective^(3, 5) (Gp23-28) + protective^(10, 15, 16)Protein: + neutralize + protective² B5R EEV + neutralize⁷ DNA: ÷neutralize (+) protective^(3, 6) (Gp42) + protective^(10, 16) Protein: ÷neutralize + protective^(2, 3)The column “antibody effect” summarizes results from referencesdescribing the effect of an antibody reactive with the named antigeneither in in vitro neutralization assays or by in vivo challengingassays to measure protectiveness. The column “immunization/vaccinationeffect” summarizes results from references where the antigen has beeninjected into animals either in protein form or as DNA. The neutralizingeffect is analyzed by assessing the neutralization titer of the injectedanimals and the protective effect by challenging theimmunized/vaccinated animals with vaccinia virus.

-   1. Demkowicz et al. 1992, J. Virol. 66:386-98.-   2. Fogg et al. 2004, J. Virol. 78:10230-7. This reference also    describes increased protection when immunization was performed with    the following protein combinations    B5R+A33R+L1R>A33R+L1R>A33R+B5R>B5R+L1R-   3. Galmiche et al, 1999, Virology 254:71-80.-   4. Gordon et al. 1991, Virology 181:671-86-   5. Hooper et al. 2000, Virology 266:329-39. This reference also    describes increased protection when vaccination was performed with    both L1R and A33R encoding DNA.-   6. Hooper et al. 2003, Virology 306:181-95. This reference also    describes increased protection when vaccination was performed with    the following DNA combinations: B5R+A33R+L1R+A27L and B5R+A27L,    where the first combination showed better protection than the second    combiniation.-   7. Law et al. 2001, Virology 280:132-42.-   8. Lai et al. 1991, J. Virol. 65:5631-5.-   9. Lin et al. 2000, J. Virol. 74:3353-3365.-   10. Lustig et al 2005, J. Virol. 79:13454-13462. This reference also    shows enhanced protection when monoclonal antibodies against L1R,    A33R and B5R were combined.-   11. Ramirez et al. 2002, J. Gen. Virol. 83:1059-1067.-   12. Rodriguez et al. 1985, J. Virol. 56:482-488.-   13. Wallengren et al. 2001, Virology 290:143-52.-   14. Wolffe et al. 1995, Virology 211:53-63.-   15. U.S. Pat. No. 6,451,309 illustrates increased protection when    monoclonal antibodies against L1R and A33R were combined. Further,    L1R and A33R mAbs combined with at least one mAb directed against    H3L, D8L, B5R, A27L and A17L is suggested, but there is no evidence    of the effect of such a combination.-   16. WO 03/068151 suggests individual or combinations of fully human    antibodies which binds an EEV protein, in particular B5R, A33R or    B7R, where B7R is a variola ortholog of B5R and shares 92.7%    identity with it. The application does not contain any evidence of    the neutralizing or protective effect of such compositions.

Some of the studies cited in table 1 have revealed that protectionagainst virus challenge is generally increased when protein/DNAcombinations targeting both IMV and EEV virion proteins are used forimmunization/vaccination (ref. 2, 5, 6 and 10). Similarly, U.S. Pat. No.6,451,309 illustrated that the combination anti-L1R and anti-A33R mAbsadministered to mice prior to a vaccinia virus challenge had anincreased protective effect compared to the individual mAbs. Thiscorrelates with early observations that vaccination with inactivated IMVparticles elicited antibody responses, but did not confer protection tovirus challenge in animal experiments (Boulter and Appleyard, 1973,Prog. Med Virol. 16, 86-108).

The fact that a combination of antibodies is better than a singlemonoclonal antibody is further supported by the observations by Gordonet al. 1991, Virology 181:671-86, where it was shown that when comparingthe neutralizing capability of a single mAb with an anti-VV envelopeserum which has been purified with respect to the same antigenspecificity as the mAb, or a non-purified polyclonal anti-VV envelopeserum, both the purified and non-purified polyclonal anti-envelope serumwas much more effective than the monoclonal antibody. Thus, both thebinding of antibodies to more than one epitope on the same antigen aswell as the binding of several antigens on different proteins is likelyto be relevant when neutralizing vaccinia virus.

DISCLOSURE OF CONTRIBUTION

The present invention provides an alternative anti-VV immunoglobulinproduct which, although it is recombinantly produced, shows reactivityto multiple antigens and epitopes of the orthopoxvirus.

DESCRIPTION OF THE INVENTION

The present invention provides a smallpox countermeasure and analternative to the serum derived VIG product, in the form of apolyclonal antibody which is capable of binding to multiple antigens andpotentially multiple epitopes on individual antigens related toorthopoxvirus infections. In contrast to serum derived VIG, a polyclonalantibody of the present invention does not contain antibody molecules,which bind to non-orthopoxvirus antigens. Thus, the polyclonal antibodyof the present invention is essentially free from immunoglobulinmolecules that do not bind to orthopoxvirus antigens. Currently,mixtures of three monoclonal antibodies produced in mice (anti-L1R,anti-A33R and anti-B5R) or mixtures of serum derived polyclonalantibodies from rabbits immunized with a particular antigen (L1R, B5R orA33R) are known (Lustig et al 2005, J. Virol. 79: 13454-13462 and U.S.Pat. No. 6,451,309). The rationale behind selecting antibodies againstexactly these three antigens was that they are directed against the IMVand EEV particles. However, since the biology of the orthopoxviruses iscomplex and not completely understood, it is highly likely that thereare other antigens in addition to these three antigens which areimportant for virus neutralization and/or protection and therebyalternative compositions may provide the same or better effects.Further, it has been shown that affinity purified serum has a greaterneutralizing effect than a single monoclonal antibody (Gordon et al.1991, Virology 181:671-86). This is likely to be due to severalantibodies binding to different epitopes on the antigen, therebyincreasing the complement activation and removal of the antigen.

The present invention provides a polyclonal anti-orthopoxvirus antibody.

Preferably, the polyclonal anti-orthopoxvirus antibody is a recombinantpolyclonal antibody (anti-orthopoxvirus rpAb), in particular an anti-VVrpAb which is directed against multiple IMV and/or EEV particle proteinsand preferably also against multiple epitopes on individual IMV/EEVproteins. Further, antibodies with reactivity against orthopoxvirusrelated regulators of complement activation (RCA) are a desiredcomponent of an anti-orthopoxvirus rpAb of the present invention.

Further, the present invention provides pharmaceutical compositionswhere the active ingredient is an anti-orthopoxvirus polyclonalantibody, as well as uses of such compositions. For example can ananti-VV rpAb of the present invention serve as replacement of thepresently used serum derived VIG and facilitate anti-variola activityfor the treatment of smallpox as an anti-terror countermeasure.

The present invention further provides screening procedures suitable forselecting a broad diversity of anti-orthopoxvirus antibodies, and inparticular procedures for mirroring the humeral immune response raisedupon challenge with an orthopoxvirus, by isolating the original V_(H)and V_(L) gene pairs from such challenged individuals, and producingantibodies maintaining this original paring.

Definitions

The term “antibody” describes a functional component of serum and isoften referred to either as a collection of molecules (antibodies orimmunoglobulin) or as one molecule (the antibody molecule orimmunoglobulin molecule). An antibody molecule is capable of binding toor reacting with a specific antigenic determinant (the antigen or theantigenic epitope), which in turn may lead to induction of immunologicaleffector mechanisms. An individual antibody molecule is usually regardedas monospecific, and a composition of antibody molecules may bemonoclonal (i.e., consisting of identical antibody molecules) orpolyclonal (i.e., consisting of different antibody molecules reactingwith the same or different epitopes on the same antigen or on distinct,different antigens). Each antibody molecule has a unique structure thatenables it to bind specifically to its corresponding antigen, and allnatural antibody molecules have the same overall basic structure of twoidentical light chains and two identical heavy chains. Antibodies arealso known collectively as immunoglobulin. The terms antibody orantibodies as used herein is used in the broadest sense and coversintact antibodies, chimeric, humanized, fully human and single chainantibodies, as well as binding fragments of antibodies, such as Fab, Fvfragments or scFv fragments, as well as multimeric forms such as dimericIgA molecules or pentavalent IgM.

The term “anti-orthopoxvirus recombinant polyclonal antibody” or“anti-orthopoxvirus rpAb” describes a composition of recombinantlyproduced diverse antibody molecules, where the individual members arecapable of binding to at least one epitope on a virus belonging to thegenus orthopoxvirus. Preferably, an anti-orthopoxvirus rpAb compositionis reactive to more than one virus species or strain belonging to thegenus orthopoxvirus. Preferably, the composition is produced from asingle manufacturing cell line, but may also be a mixture of monoclonalantibodies or any combination of an anti-orthopoxvirus rpAb compositionand one or more monoclonal antibodies. The diversity of the polyclonalantibody is located in the variable regions (V_(H) and V_(L) regions),in particular in the CDR1, CDR2 and CDR 3 regions.

The term “anti-VV recombinant polyclonal antibody” or “anti-VV rpAb”describes a composition of recombinantly produced diverse antibodymolecules, where the individual members are capable of binding to atleast one epitope on a vaccinia virus species or strain. Preferably, ananti-VV rpAb composition is reactive to at least one IMV and at leastone EEV specific antigen, where the reactivity is characterized bydistinct members reactive to either an IMV or an EEV specific antigen.Preferably, the composition is produced from a single manufacturing cellline, but it may also be a mixture of monoclonal antibodies. Thediversity of the polyclonal antibody is located in the variable regions(V_(H) and V_(L) regions), in particular in the CDR1, CDR2 and CDR 3regions.

The term “the polyclonal antibody of the present invention isessentially free from immunoglobulin molecules that do not bind toorthopoxvirus antigens” means that more than 80% of the antibodies,preferably more than 90%, more preferably more than 95% and mostpreferably more than 99%, bind to one of the orthopoxvirus antigens.

The term “cognate V_(H) and V_(L) coding pair” describes an originalpair of V_(H) and V_(L) coding sequences contained within or derivedfrom the same cell. Thus, a cognate V_(H) and V_(L) pair represents theV_(H) and V_(L) pairing originally present in the donor from which sucha cell is derived. The term “an antibody expressed from a V_(H) andV_(L) coding pair” indicates that an antibody or an antibody fragment isproduced from a vector, plasmid or similar containing the V_(H) andV_(L) coding sequence. When a cognate V_(H) and V_(L) coding pair isexpressed, either as a complete antibody or as a stable fragmentthereof, they preserve the binding affinity and specificity of theantibody originally expressed from the cell they are derived from. Alibrary of cognate pairs is also termed a repertoire or collection ofcognate pairs, and may be kept individually or pooled.

The terms “a distinct member of a recombinant polyclonal antibody”denotes an individual antibody molecule of the recombinant polyclonalantibody composition, comprising one or more stretches within thevariable regions, which are characterized by differences in the aminoacid sequence compared to the other individual members of the polyclonalprotein. These stretches are in particular located in the CDR1, CDR2 andCDR 3 regions.

The term “epitope” is commonly used to describe a site on a largermolecule (e.g. antigen) to which the antibody will bind. An antigen is asubstance that stimulates an immune response, e.g. toxin, virus,bacteria, proteins or DNA. An antigen often has more than one epitope,unless they are very small. Antibodies binding to different epitopes onthe same antigen can have varying effects on the activity of the antigenthey bind depending on the location of the epitope. An antibody bindingto an epitope in an active site of the antigen may block the function ofthe antigen completely, whereas another antibody binding at a differentepitope may have no or little effect on the activity of the antigen.Such antibodies, may however still activate complement and therebyresult in the elimination of the antigen.

The term “fully human” used for example in relation to DNA, RNA orprotein sequences describes sequences which are between 98 to 100%human.

The term “immunoglobulin” commonly is used as a collective designationof the mixture of antibodies found in blood or serum, but may also beused to designate a mixture of antibodies derived from other sources.

The term “mirrors the humeral immune response” when used in relation toa polyclonal antibody refers to an antibody composition where thenucleic acid sequences encoding the individual antibody members arederived from a donor who either has been subject to vaccination with anorthopoxvirus or who is recovering from an infection with anorthopoxvirus. In order to mirror the affinity and specificity ofantibodies raised in a donor upon challenge, the sequences encoding thevariable heavy chain (V_(H)) and the variable light chain (V_(L)) shouldbe maintained in the gene pairs or combinations originally present inthe donor (cognate pairs) when they are isolated. In order to mirror thediversity of a humeral immune response in a donor all the sequencesencoding antibodies which bind to an orthopoxvirus are selected based ona screening procedure. The isolated sequences are analyzed with respectto diversity of the variable regions, in particular the CDR regions, butalso with respect to the V_(H) and V_(L) family. Based on these analysesa population of cognate pairs representing the overall diversity of theorthopoxvirus binding antibodies are selected. Such a polyclonalantibody typically have at least 8, 10, 20, 30, 40, 50, 100, 1000 or 10⁴distinct members.

The term “orthopoxvirus” refers to a virus species or strain belongingto the genus orthopoxvirus. Known viruses include Buffalopox, Californiavole pox, camelpox, cowpox, ectromelia, monkeypox, rabbitpox, raccoonpox, tatera pox, Uasin Gishu pox, vaccinia, variola, and vole pox virus.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient.

The term “unit dose form” refers to a ready-to-administer formcomprising a therapeutically effective amount of the active ingredient.

The term “polyclonal antibody” describes a composition of different(diverse) antibody molecules which is capable of binding to or reactingwith several different specific antigenic determinants on the same or ondifferent antigens. Usually, the variability of a polyclonal antibody islocated in the so-called variable regions of the polyclonal antibody, inparticular in the CDR regions. In the present invention a polyclonalantibody may either be produced in one pot from a polyclonal cell line,or it may be a mixture of different polyclonal antibodies. A mixture ofmonoclonal antibodies is not as such considered a polyclonal antibody,since they are produced in individual batches and not necessarily formthe same cell line which will result in e.g. post translationalmodification differences. However, if a mixture of monoclonal antibodiesprovide the same antigen/epitope coverage as a polyclonal antibody ofthe present invention it will be considered as an equivalent of thepolyclonal antibody. When stating that a member of a polyclonal antibodybinds to an antigen, it is herein meant a binding having bindingconstant that is below 100 nM, preferably below 10 nM, even morepreferred below 1 nM.

The term “recombinant antibody” is used to describe an antibody moleculeor several molecules that is/are expressed from a cell or cell linetransfected with an expression vector comprising the coding sequence ofthe antibody which is not naturally associated with the cell. If theantibody molecules in a recombinant antibody composition are diverse ordifferent, the term “recombinant polyclonal antibody” or “rpAb” appliesin accordance with the definition of a polyclonal antibody.

The term “recombinant polyclonal cell line” or “polyclonal cell line”refers to a mixture/population of protein expressing cells that aretransfected with a repertoire of variant nucleic acid sequences (e.g. arepertoire of antibody encoding nucleic acid sequences). Preferably, thetransfection is performed such that the individual cells, which togetherconstitute the recombinant polyclonal cell line, each carry atranscriptionally active copy of a single distinct nucleic acid sequenceof interest, which encodes one member of the recombinant polyclonalantibody of interest. Even more preferred, only a single copy of thedistinct nucleic acid sequence is integrated at a specific site in thegenome. The cells constituting the recombinant polyclonal cell line areselected for their ability to retain the integrated copy of the distinctnucleic acid sequence of interest, for example by antibiotic selection.Cells which can constitute such a polyclonal cell line can be forexample bacteria, fungi, eukaryotic cells, such as yeast, insect cells,plant cells or mammalian cells, especially immortal mammalian cell linessuch as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0cells, NS0), NIH 3T3, YB2/0 and immortalized human cells, such as HeLacells, HEK 293 cells, or PER.C6.

The terms “sequences encoding V_(H) and V_(L) pairs” or “V_(H) and V_(L)encoding sequence pairs” indicate nucleic acid molecules, where eachmolecule comprise a sequence that code for the expression of a variableheavy chain and a variable light chain, such that these can be expressedas a pair from the nucleic acid molecule if suitable promoter and/orIRES regions are present and operably linked to the sequences. Thenucleic acid molecule may also code for part of the constant regions orthe complete constant region of the heavy chain and/or the light chain,allowing for the expression of a Fab fragment, a full-length antibody orother antibody fragments if suitable promoter and/or IRES regions arepresent and operably linked to the sequences.

A recombinant polyclonal antibody is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant, e.g. prevents or attenuates anorthopoxvirus infection in an animal or human.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of amino acid sequences of known orthopoxvirus relatedregulators of complement activation (RCA), including a consensussequence.

FIG. 2: Schematic outline of the multiplex overlap-extension RT-PCR (A)and the cloning steps (B). (A) Two sets of primers, CH+VH 1-8 andVK1-6+CK1, specific for V_(H) and V_(κ) gene families, respectively,were used for the first PCR step. A homologous region between the V_(H)or V_(κ) primers results in the generation of an overlap PCR product. Inthe second step this product is amplified in the nested PCR. The primersalso include recognition sites for restriction enzymes that facilitatecloning. (B) The generated cognate linked V_(H) and V_(κ) coding pairsare pooled and inserted into a Fab expression vector by the use of theflanking XhoI and NotI restriction sites. Subsequently a bi-directionalpromoter is inserted into the AscI-NneI restriction site between thelinked V_(H) and V_(κ) coding sequences to facilitate Fab expression.PCR primers used are indicated by horizontal arrows. CH1: heavy chainconstant domain 1, CL: constant domain, LC: light chain; Ab: antibody;P1-P2: bi-directional promoters.

FIG. 3: Schematic presentations of suitable Fab expression vectors. A)Shows JSK301, an E. coli Fab expression vector with NotI/XhoIrestriction sites for insertion of linked V_(H) and V_(L) coding pairs.The vector comprises the following elements: Amp and Amp pro=ampicillinresistance gene and its promoter. pUC19 Ori=origin of replication. HumanCH1=sequence encoding human immunoglobulin gamma 1 heavy chain domain 1.Stuffer=an irrelevant sequence insert which is cut out upon insertion ofthe overlap extension fragments. tac P and lac Z=bacterial promoterswhich can be excised at the NheI and AscI restriction sites andsubstituted with other promoter pairs. B) Shows a mammalian Fabexpression vector with NotI/XhoI restriction sites for insertion oflinked V_(H) and V_(L) coding pairs. The vector comprises the followingelements: Amp and Amp pro=ampicillin resistance gene and its promoter.pUC origin=origin of replication. Rabbit B-globin A=rabbit beta-globinpolyA sequence. IgG1 CH1=sequence encoding human immunoglobulin gamma 1heavy chain domain 1. VH=sequence encoding variable heavy chain. HCleader=A genomic human heavy chain leader. P1=mammalian promoter drivingthe expression of the light chain. P2=mammalian promoter driving theexpression of the heavy chain. Kappa leader=A murine genomic kappa chainleader. LC=light chain encoding sequence. SV40 term=Simian virus 40terminator sequence. Neo=Neomycin resistance gene. SV40 PolyA=Simianvirus 40 poly A signal sequence.

FIG. 4: A schematic presentation of a mammalian full-length antibodyexpression vector 00-VP-530. The vector comprises the followingelements: Amp and Amp pro ampicillin resistance gene and its promoter.pUC origen=pUC origin of replication. P1=mammalian promoter driving theexpression of the light chain. P2=mammaliaa promoter driving theexpression of the heavy chain. Leader IGHV=genomic human heavy chainleader. VH=heavy chain variable region encoding sequence. IgG1 Sequenceencoding for genomic immunoglobulin isotype G1 heavy chain constantregion. Rabbit B-globin A=rabbit beta-globin polyA sequence. Kappaleader sequence encoding for murine kappa leader. LC=Sequence encodinglight chain encoding sequence. SV40 term=Simian virus 40 terminatorsequence. FRT=A Flp recognition target site. Neo=neomycin resistancegene. SV40 poly A=Simian virus 40 poly A signal sequence

FIG. 5: Representative Western blots of the identified antigen groups.Virus particles or recombinant antigens were separated by SDS-PAGE, andblotted. The Western blots were probed with the indicated antibodyidentified by its clone number. The identified antigens are indicatedabove each blot. The antigen group assigned A did not give anydetectable band in Western blot. M indicates the marker. The size of themarker is 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 80 kDa, 100 kDa, 120kDa.

FIG. 6: Flow chart illustrating the antibody screening process. The Fabfragments or full length antibodies (Abs) were screened againstinactivated vaccinia virus strains and recombinant vaccinia virusassociated protein in a first round of screening generating primaryhits. The primary hits were then subjected to a second round ofscreening. Generally the first round of screening was performed usingFLISA and the second round of screening was performed with FLISA and/orELISA. The figure exemplifies EEV or non-particle associated proteins asrecombinant vaccinia virus associated proteins. However recombinant IMVspecific proteins such as L1R can also be used.

FIG. 7: Anti-vaccinia virus reactivity in 10 donors (indicated by D-001to D-011). Antibody titers against recombinant antigens A27L, A33R, B5R,L1R, and VCP and virus particles from different strains (Lister, IHD-W,IHD-J) were determined as the minimum dilution producing a signal 4-foldabove background in ELISA.

FIG. 8: Anti-vaccinia virus reactivity of the Mini-H and Mini-Vcompositions compared to the serum derived SymVIG. The binding wasdetected by ELISA using inactivated Lister strain particles as antigenand the negative control was anti-RhD polyclonal antibody.

FIG. 9: In vivo inhibition of vaccinia virus replication by Mini-V inthe mouse tail lesion vaccinia virus replication model. The indicatedamount of Mini-V or SymVIG was injected intraperitoneally 24 hours afterviral challenge. Either the Lister strain (dashed) or the NYCBOH strain(solid) was used for challenge. Results are percentages of lesionscompared with the control group treated with an irrelevant recombinantpolyclonal antibody (anti-RhD rpAb). The small molecule drug Cidofovirwas included as positive control and administered intramuscularly(i.m.).

FIG. 10: IEX profile of anti-VV rpAb compound. The individual clonesidentified by the clone numbers given in Table 5 are assigned to theindividual peaks. Cell lines expressing 02-113 and 02-225 were includedin the polyclonal cell line but were not found in the anti-VV rpAb.

FIG. 11: Vaccinia virus binding of the three anti-VV rpAb batches andthe two VIG products, SymVIG and Cangene VIG (VIG). The binding wastested in ELISA against the antigens indicated at the top of each plot.

FIG. 12: Sym002 anti-VV rpAb (SYM002) inhibits vaccinia virusreplication in vivo. Prophylactic (lower panel) or therapeutic (toppanel) intramuscular administered Sym002 anti-VV rpAb inhibits both theLister and NYCBOH vaccinia virus replication in vivo as assayed by themouse tail pock model. All the four data sets showed an approximately300-fold increased specific activity of Sym002 anti-VV rpAb as comparedto VIG (Cangene).

FIG. 13: Sym002 mix and Sym002 anti-VV rpAb (SYM002) have identicalanti-viral potency in the mouse tail lesion model. Eight mice in eachgroup were challenged with vaccinia virus, NYCOBH strain 24 hours priorto injection I.P. injection of the indicated amount of antibodies.

FIG. 14: Affinities of antibodies reactive against B5R, A33R, A27L, orVCP determined by surface plasmon resonance using a BIAcore 2000. Theobtained affinities are in the nanomolar range indicating affinitymaturated antibodies.

DETAILED DESCRIPTION OF THE INVENTION Target Antigens and PolyclonalAntibody Compositions

A polyclonal antibody of the present invention is composed of a numberof distinct antibody molecules in the same composition. Each molecule isselected based on its ability to bind an orthopoxvirus associatedantigen. Preferably, the distinct members of a polyclonalanti-orthopoxvirus antibody of the present invention are capable ofbinding at least three orthopoxvirus related antigens in union. Further,it is preferred that each distinct antibody of the polyclonal antibodybinds an epitope which is not bound by any of the other members of thepolyclonal antibody. An antibody of the polyclonal antibody compositionmay bind an epitope which overlaps epitopes of other distinct antibodiesof the composition and still be considered a distinct antibody. Anadditional feature of an anti-orthopoxvirus polyclonal antibody of thepresent invention is the capability of binding at least two distinctepitopes on the same orthopoxvirus related antigen, therebysupplementing the binding to at least three different orthopoxvirusassociated antigens. Such a polyclonal antibody is, hence, composed ofat least 4 distinct antibody members. A polyclonal antibody of thepresent invention comprises binding reactivity corresponding to thecompiled binding reactivity of the distinct antibody moleculesconstituting the polyclonal antibody composition. Preferably, apolyclonal antibody of the present invention is produced as a singlebatch or a few batches from a polyclonal cell line which is notnaturally expressing antibody molecules (also termed a recombinantpolyclonal antibody). One of the advantages of producing a recombinantpolyclonal antibody compared to mixing monoclonal antibodies, is theability to produce an principally unlimited number of distinct antibodymolecules at the same time (at a cost similar to that of producing asingle monoclonal antibody). Thus, it is possible to include antibodieswith reactivity towards a large number of orthopoxvirus associatedantigens, known as well as unknown, without increasing the cost of theend product significantly. In particular with a target as complex as theorthopoxviruses where the biology is not completely understood,individual antibodies which have not been shown to neutralize or protectagainst orthopoxviruses alone, may when combined with other antibodiesinduce a synergistic effect. Thus, it can be an advantage to includedistinct antibodies in a polyclonal antibody composition, where the onlycriterion is that the individual antibody binds to an orthopoxvirusantigen.

One way to acquire potentially relevant antibodies that bindorthopoxvirus target antigens which have not been verified as relevantantigens, but none the less may be so, is to generate a polyclonalantibody composition which is composed of individual antibodies raisedby the immune response of a donor which has been vaccinated or infectedwith an orthopoxvirus (full immune response). In addition to broadlyobtaining antibodies derived from a full immune response againstothopoxviruses, a positive selection for antibodies binding to antigensthat are likely to be of particular relevance in the protection,neutralization, and/or elimination of orthopoxvirus infections or in theprotection against adverse effects from vaccina virus vaccination, canbe performed. Further, if antibodies to a particular antigen, which isknown to be of relevance in the protection, neutralization and/orelimination of orthopoxvirus are not identified in the full immuneresponse of the donor, such antibodies may be raised byimmunization/vaccination of a donor with that particular antigen(selected immune response). Generally, neutralization is assessed by invitro neutralization assays such as plaque reduction neutralizationassays (PRNT assay) using either IMV or EEV preparations, by cometassay, EEV neutralization assay (EEV specific) (Law et al. 2001,Virology 280:132-42) or by flow cytometric detection of greenfluorescent protein (Earl et al. 2003 J. Virol. 77: 10684-88).Protection is generally assessed by in vivo challenging experiments suchas the mouse tail lesion model, lethal dose challenge or footpadmeasurements. The in vivo challenging experiments can either beperformed in a prophylactic fashion, where the antibodies areadministered prior to the viral challenge or as a treatment, where theantibodies are administered after viral challenge or as a combination ofboth.

A polyclonal antibody composition of the present invention can becomposed of antibodies capable of binding an orthopoxvirus antigen whichis not necessarily known, but where the antibodies are acquired from afull immune response to an orthopoxvirus, e.g. by obtaining nucleic acidsequences encoding the distinct antibodies from one or more donorsvaccinated with an orthopoxvirus, or recovering from an orthopoxvirusinfection. Secondly, antibodies from the same full immune response,which have been selected based on their ability to bind a particularantigen and/or epitope, can be included in a polyclonal antibody of thepresent invention. Thirdly, distinct antibodies encoded from V_(H) andV_(L) pairs obtained from one or more donors which have beenimmunized/vaccinated with a particular orthopoxvirus related antigenthereby raising a “selected” immune response in these donors, can beincluded in a polyclonal antibody composition of the present invention.Thus, antibodies derived by any of the mentioned techniques in thepresent invention may be combined into a single polyclonal antibody.Preferably the nucleic acids encoding the antibodies of the presentinvention are obtained from human donors and the antibodies produced arefully human antibodies.

The motivation behind the polyclonal antibody compositions of thepresent invention is: if a donor immunized or infected with anorthopoxvirus, raises an humeral immune response against an antigen,these antibodies are likely at least to some extend to contribute toviral clearance, and thereby qualify for inclusion in a polyclonalantibody product.

One embodiment of the present invention is an anti-orthopoxvirus rpAbwherein the composition of distinct antibody members mirrors the humeralimmune response with respect to diversity, affinity and specificityagainst antigens associated with one or more orthopoxviruses, inparticular vaccinia virus, variola virus and/or monkeypox virus.Preferably, the mirror of the humeral response is established byensuring that one or more of the following are fulfilled i) the nucleicacid sequences coding for the V_(H) and V_(L) regions of the individualantibody members in such an anti-orthopoxvirsus rpAb are derived from adonor(s) who has raised a humeral immune response against anorthopoxvirus, for example following vaccination with vaccinia virus oran orthopox virus infection from which the donor is recovering; ii) theV_(H) and V_(L) coding sequences are isolated from the donor(s) suchthat the original pairing of the V_(H) and V_(L) coding sequencespresent in the donor(s) is maintained, iii) the V_(H) and V_(L) pairs,coding for the individual members of the rpAb, are selected such thatthe CDR regions are as diverse as possible; or iv) the specificity ofthe individual members of the anti-orthopoxvirus rpAb are selected suchthat the antibody composition collectively binds antigens that elicitsignificant antibody responses in mammals. Preferably, the antibodycomposition collectively binds antigens which produce significantantibody titers in a serum sample from said donor(s).

The antigens of relevance to the present invention are any orthopoxvirusderived protein, polypeptide or nucleic acid, towards which a humeralimmune response or a selected immune response can be raised. Therelevant antigens can be selected from viral proteins presented at thesurface of IMV and/or EEV particles. At least twelve different viralproteins, (here referred to by the vaccinia virus variants) A14.5L,E10R, I5L, A13L, A27L, A17L, L1R, L5R, DBL, H3L, A14L and A17L, areinserted in the IMV outer membrane and may be relevant antigensaccording to the present invention. Further, at least six otherproteins, A33R, A34R, F13L, B5R, A56R, F12L and A36R, are present at thesurface membrane of the EEV particle and may likewise be relevantantigens according to the present invention.

In an additional embodiment of the present invention theanti-orthopoxvirus rpAb comprises binding reactivity against antigensselected from the group of viral proteins associated with IMV and/or EEVparticles, in particular proteins presented on the surface of theseparticles. In a preferred embodiment of the present invention theanti-orthopoxvirus rpAb comprises binding reactivity against antigensselected among the viral proteins A27L, A17L, D8L and H3L, and againstantigens selected among the viral proteins A33R and B5R. Additionally,the first group can constitute the viral proteins L1R and the secondgroup can constitute the viral protein A56R.

Further, additional orthopoxvirus proteins have shown immunoreactivityin humans, macaques and/or mice following vaccination with vacciniavirus. These include the vaccinia virus ortholog A10L, A11R, D13L, H5R,A26L, E3L, L4R, H7R, P4A and A4L (Davies et al. 2005, PNAS 102: 547-552and Demkowicz et al. 1992, J. Virol. 66:386-98), and are also consideredas potentially relevant antigens to which individual members of apolyclonal antibody according to the present invention can bind. In afurther embodiment of the present invention, the polyclonal antibodycomprises binding reactivity against one or more of the antigensselected from the following group of viral proteins, represented as thevaccinia virus ortholog A14.5L, E10R, ISL, A13L, A27L, A17L, L1R, D8L,H3L, A14L, A17L, A33R, A34R, F13L, B5R, A56R, F12L, A36R, A10L, A11R,D13L, H5R, A26L, E3L, L4R, H7R, P4A and A4L.

The viral proteins mentioned above are, however, not the only viralproteins with potential relevance in the protection, neutralizationand/or elimination of orthopoxvirus infections or prevention of adverseeffects due to vaccination with vaccinia virus. The orthopoxvirusesencode regulators of complement activation (RCA), that contain fourtandem short consensus repeats (SCRs), allowing them to evade theconsequences of complement activation in the host (reviewed in Mullicket al. 2003, Trends Immunol. 24:500-7). Several RCA proteins have beenidentified in the group of orthopoxviruses, namely vaccinia viruscomplement control protein (VCP), smallpox inhibitor of complementenzyme (SPICE) and inflammatory modulatory protein (IMP), from vacciniavirus, variola virus and cowpox, respectively. Certain monkeypox viralstrains also have an ortholog of the VCP, which may be responsible forthe violence of these particular strains, compared to other monkeypoxstrains (Chen et al. 2005, Virology 340:46-63). Further, a sequencerelating to a VCP protein from camelpox virus has also been identifiedunder NCBI accession number AAL73730. FIG. 1 shows an alignment of thementioned RCA proteins, including an orthopoxvirus RCA protein consensussequence. US 2005/0129700 describes the generation of anti-VCP andanti-SPICE antibodies, preferably with reactivity to both antigens. Themonoclonal antibodies are used to pre-inject animals which are subjectedto vaccinia virus vaccination, there are however no indication whetherthe antibodies are protective or not. Further, a series of monoclonalmouse antibodies against VCP has been generated in order to map the SCRdomains involved in abolishing complement-enhanced neutralization.Antibodies binding to the SCR2, SCR4 or the junction between the SCR3and 4 domains blocked the interaction of VCP with complement (Isaacs etal. 2003, J. Virol. 77:8256-62).

In further embodiments of the present invention the above mentionedpolyclonal antibody compositions additionally comprise bindingreactivity against an RCA encoded by an orthopoxvirus. In particular apolyclonal anti-orthopoxvirus composition comprising binding reactivityagainst IMV, EEV and RCA specific antigens is desired. A furtheranti-orthopoxvirus polyclonal antibody of the present inventioncomprises binding reactivity against antigens selected among the viralproteins (vaccinia virus orthologs) A27L, A17L, D8L, H3L, A33R, B5R andVCP. Additionally, the group can constitute the viral proteins L1Rand/or A56R. In any of the embodiments of the present invention relatingto RCA specific antibody members, the RCA binding specificity ispreferably directed against a protein selected from the group VCP,SPICE, IMP, MPXV-VCP and CMLV-VCP. In a preferred embodiment the rpAbcompositions of the present invention comprises individual members withbinding reactivity against the orthopoxvirus RCA protein consensussequence. In an even more preferred embodiment the RCA related bindingreactivity is directed to an epitope located in the SCR2, SCR4 and/orthe junction between the SCR3 and 4 domains of one of the mentioned RCAproteins or the RCA protein consensus sequence. Preferably the VCPreactivity is directed against the SCR2, SCR4 and/or the junctionbetween the SCR3 and 4 domains.

The present invention has identified a series of V_(H) and V_(L) pairsthat can be expressed as full-length antibodies, Fab fragment or otherantibody fragments that have binding specificity to an vaccinia virusassociated antigen. The specific V_(H) and V_(L) pairs are identified byclone number in Table 5 in Example 2. An antibody containing a V_(H) andV_(L) pair as identified in Table 5 is preferably a fully humanantibody. However, if desired chimeric antibodies may also be produced.

A preferred anti-orthopoxvirus recombinant polyclonal antibody of thepresent invention is composed of distinct members comprising heavy chainand light chain CDR1, CDR2 and CDR3 regions selected from the group ofV_(H) and V_(L) pairs listed in Table 5. Preferably, the CDR regions aremaintained in the pairing indicated in Table 5 and inserted into adesired framework. Alternatively CDR regions from the heavy chain (CDRH)of a first clone are combined with the CDR regions from the light chain(CDRL) of a second clone (scrambling of V_(H) and V_(L) pairs). The CDRregions may also be scrambled within the light chain or heavy chain, forexample by combining the CDRL1 region from a first clone with the CDRL2and CDRL3 region from a second clone. Such scrambling is preferablyperformed among clones that bind the same antigen. The CDR regions ofthe present invention may also be subjected to affinity maturation, e.g.by point mutations.

An even more preferred anti-orthopoxvirus recombinant polyclonalantibody of the present invention is comprised of distinct members withheavy chain and light chain CDR1, CDR2 and CDR3 regions corresponding toclone numbers 02-029, 02-037, 02-058, 02-086, 02-147, 02-186, 02-188,02-195, 02-197, 02-203, 02-211, 02-229, 02-235, 02-286, 02-295, 02-303,02-339, 02-461, 02-482, 02-488, 02-526, 02-551, 02-586, 02-589, 02-607and 02-633.

In a further embodiment, the above composition, comprising 26 individualmembers, additionally comprise the following two distinct members withheavy chain and light chain CDR1, CDR2 and CDR3 regions corresponding toclone numbers 02-113 and 02-225.

A further aspect of the present invention is the individual antibodies,identified by the method of the present invention, which bind previouslyunidentified epitopes of an orthopoxvirus, in particular vaccinia virusand/or variola virus.

In addition to the antigens mentioned above, individual antibodies withbinding specificity towards unidentified antigens can be identified byfor example Western blot analysis using inactivated orthopoxvirusparticles as antigen source. These unidentified antigens may correspondto known antigens, but they may also correspond to unknown antigens. Theidentity of an antigen toward which an antibody of the present inventionbinds, can be assessed by analyzing binding specificity of theidentified antibodies to recombinant proteins of known antigens.Alternatively, competition assays against antibodies with a knownspecificity can be performed. Such competition assays does, however, notexclude that the identified antibody binds to the same antigen as theknown antibody, since it may bind to a different epitope.

The present invention has identified antibodies against the followingantigens from the Lister strain by Western blotting: B (˜82 kDa), C(35-40 kDa), D (Three band appearance ˜65, ˜72, ˜95 kDa), E (32-35 kDA),G (Three band appearance 80 kDa, 60 kDa, 31-33 kDa), HI (˜35 Da), J(35-38 kDa) and L (<20 kDa). Antibodies against the known antigen VCP,B5R, A27L and A33R have also been verified by Western blotting. By usingin vitro translated proteins the HI antigen was shown to correspond toantigen H3L, the E antigen to D8L, the D antigen to A56R, the remainingantigens does not correspond to VCP, B5R, A27L, A33R or H3L, D8L, A56Rand the antibodies binding to these unidentified antigens maypotentially bind to previously unknown orthopoxvirus antigens orepitopes.

An embodiment of the present invention is an antibody binding antigen Bof the Lister strain at the same epitope as an antibody comprising threeheavy chain CDRs and three light-chain CDRs derivable from clone nr.02-037, 02-089 and/or 02-058.

An embodiment of the present invention is an antibody binding antigen Cof the Lister strain at the same epitope as an antibody comprising threeheavy chain CDRs and three light-chain CDRs derivable from clone nr.02-243.

An embodiment of the present invention is an antibody binding antigen Dor A56R of the Lister strain at the same epitope as an antibodycomprising three heavy chain CDRs and three light-chain CDRs derivablefrom clone nr. 02-628, 02-431,002-516 and/or 02-551.

An embodiment of the present invention is an antibody binding antigen Eor D8L of the Lister strain at the same epitope as an antibodycomprising three heavy chain CDRs and three light-chain CDRs derivablefrom clone nr. 02-339.

An embodiment of the present invention is an antibody binding antigen Gof the Lister strain at the same epitope as an antibody comprising threeheavy chain CDRs and three light-chain CDRs derivable from clone nr.02-147.

An embodiment of the present invention is an antibody binding antigen Jof the

Lister strain at the same epitope as an antibody comprising three heavychain CDRs and three light-chain CDRs derivable from clone nr. 02-640and/or 02-633.

An embodiment of the present invention is an antibody binding antigen Lof the

Lister strain at the same epitope as an antibody comprising three heavychain CDRs and three light-chain CDRs derivable from clone nr. 02-589,02-156, and/or 02-225.

Isolation and Selection of Variable Heavy Chain and Variable Light ChainCoding Pairs

The process of generating an anti-orthopoxvirus recombinant polyclonalantibody composition involves the isolation of sequences coding forvariable heavy chains (V_(H)) and variable light chains (V_(L)) from asuitable source, thereby generating a repertoire of V_(H) and V_(L)coding pairs. Generally, a suitable source for obtaining V_(H) and V_(L)coding sequences are lymphocyte containing cell fractions such as blood,spleen or bone marrow samples from an animal or humanimmunized/vaccinated with an orthopoxvirus strain or proteins or DNAderived from such a strain. Preferably, lymphocyte containing fractionsare collected from humans or transgenic animals with humanimmunoglobulin genes, which have been vaccinated with a vaccinia virusstrain, such a strain can for example be selected from a group ofstrains comprising Connaught, IHD-J, IHD-W, Brighton, WT, Lister,NYCBOH, Copenhagen, Ankara, Dairen I, L-IPV, LC16MO, LIVP, Tian Tan, WR65-16, and Wyeth or proteins or DNA derived from such a strain. Patientsrecovering from an infection with an orthopoxvirus strain can also beused as source for the V_(H) and V_(L) gene isolation. The collectedlymphocyte containing cell fraction may be enriched further to obtain aparticular lymphocyte population, e.g. cells of the B lymphocytelineage. Preferably, the enrichment is performed using magnetic beadcell sorting (MACS) and/or fluorescence activated cell sorting (FACS),taking advantage of lineage-specific cell surface marker proteins forexample for B cells and/or plasma cells. Preferably, the lymphocytecontaining cell fraction is enriched with respect to B cells and/orplasma cells. Even more preferred cells with high CD19 and CD38expression and intermediate CD45 expression are isolated from blood.These cells are sometimes termed circulating plasma cells, early plasmacells or plasmablasts, for ease, they are just termed plasma cells inthe present invention.

The isolation of V_(H) and V_(L) coding sequences can either beperformed in the classical way where the V_(H) and V_(L) codingsequences are combined randomly in a vector to generate a combinatoriallibrary of V_(H) and V_(L) coding sequences pairs. However, in thepresent invention it is preferred to mirror the diversity, affinity andspecificity of the antibodies produced in a humeral immune response uponchallenge with an orthopoxvirus. This involves the maintenance of theV_(H) and V_(L) pairing originally present in the donor, therebygenerating a repertoire of sequence pairs where each pair encodes avariable heavy chain (V_(H)) and a variable light chain (V_(L))corresponding to a V_(H) and V_(L) pair originally present in anantibody produced by the donor from which the sequences are isolated.This is also termed a cognate pair of V_(H) and V_(L) encoding sequencesand the antibody is termed a cognate antibody. Preferably, the V_(H) andV_(L) coding pairs of the present invention, combinatorial or cognate,are obtained from human donors, and therefore the sequences arecompletely human.

There are several different approaches for the generation of cognatepairs of V_(H) and V_(L) encoding sequences, one approach involves theamplification and isolation of V_(H) and V_(L) encoding sequences fromsingle cells sorted out from a lymphocyte-containing cell fraction. TheV_(H) and V_(L) encoding sequences may be amplified separately andpaired in a second step or they may be paired during the amplification(Coronella et al. 2000 Nucleic Acids Res. 28: E85; Babcook et al 1996PNAS 93: 7843-7848 and WO 05/042774). An alternative approach involvesin-cell amplification and pairing of the V_(H) and V_(L) encodingsequences (Embleton et al. 1992. Nucleic Acids Res. 20: 3831-3837;Chapal et al. 1997 BioTechniques 23: 518-524). In order to obtain arepertoire of V_(H) and V_(L) encoding sequence pairs which resemble thediversity of V_(H) and V_(L) sequence pairs in the donor, ahigh-throughput method with as little scrambling (random combination) ofthe V_(H) and V_(L) pairs as possible, is preferred, e.g. as describedin WO 05/042774 (hereby incorporated by reference).

In a preferred embodiment of the present invention a repertoire of V_(H)and V_(L) coding pairs, where the member pairs mirror the gene pairsresponsible for the humeral immune response upon challenge with anorthopoxvirus, is generated according to a method comprising the stepsi) providing a lymphocyte-containing cell fraction from a donorvaccinated with an orthopoxvirus or recovering from an orthopoxvirusinfection; ii) optionally enriching B cells or plasma cells from saidcell fraction; iii) obtaining a population of isolated single cells,comprising distributing cells from said cell fraction individually intoa plurality of vessels; iv) amplifying and effecting linkage of theV_(H) and V_(L) coding pairs, in a multiplex overlap extension RT-PCRprocedure, using a template derived from said isolated single cells andv) optionally performing a nested PCR of the linked V_(H) and V_(L)coding pairs. Preferably the isolated cognate V_(H) and V_(L) codingpairs are subjected to a screening procedure as described below.

Once the V_(H) and V_(L) sequence pairs have been generated a screeningprocedure to identify sequences encoding V_(H) and V_(L) pairs withbinding reactivity towards an orthopoxvirus associated antigen isperformed. If the V_(H) and V_(L) sequence pairs are combinatorial aphage display procedure can be applied to enrich for V_(H) and V_(L)pairs coding for antibody fragments binding to orthopoxvirus prior toscreening.

in order to mirror the diversity, affinity and specificity of theantibodies produced in a humeral immune response upon challenge with anorthopoxvirus, the present invention has developed a screening procedurefor the cognate pairs, in order to obtain the broadest diversitypossible. For screening purposes the repertoire of cognate V_(H) andV_(L) coding pairs are expressed individually either as antibodyfragments (e.g. scFv or Fab) or as full-length antibodies using either abacterial or mammalian screening vector transfected into a suitable hostcell. The repertoire of Fabs/antibodies is first screened for reactivityto one or more orthopoxvirus strains. In parallel the Fabs/antibodies,are screened against selected antigens. These antigens are selectedbased on the knowledge of the orthopoxvirus biology and the expectedneutralizing and/or protective effect antibodies capable of binding tothese antigens potentially can provide. This screening procedure canlikewise be applied to a combinatorial phage display library.

In an embodiment of the present invention the screening procedure forselecting V_(H) and V_(L) sequence pairs capable of encoding a broaddiversity of anti-orthopoxvirus antibodies is performed as follows: anantibody or antibody fragments is expressed from a host cell transfectedwith a screening vector containing a distinct member of the repertoireof V_(H) and V_(L) coding pairs. The antibody or antibody fragment isscreened against at least two different vaccinia virus strains inconjunction with a parallel screening against one or more of thefollowing antigens A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP bycontacting the antibody or fragment with these strains/antigens. Thisprocess is performed for each member of the repertoire of V_(H) andV_(L) coding pairs, and sequences encoding V_(H) and V_(L) pairs thatbind to either the whole virus and/or one of the specific antigens areselected from the host cells containing them (also termed clones).Preferably a second screening is performed, in order to ensure that noneof the selected sequences encode false positives. In the secondscreening all the vaccinia virus/antigen binding V_(H) and V_(L) pairsidentified in the first screening are screened again against both thevirus strains and the selected antigens. The screening procedure isillustrated in FIG. 6, exemplified with some of the antigens mentionedabove. Generally, immunological assays are suitable for the screeningperformed in the present invention. Such assays are well know in the artand constitute for example ELISPOTS, ELISA, FLISA, membrane assays (e.g.Western blots), arrays on filters, and FACS. The assays can either beperformed without any prior enrichment steps, utilizing polypeptidesproduced from the sequences encoding the V_(H) and V_(L) pairs. In theevent that the repertoire of V_(H) and V_(L) coding pairs are cognatepairs no enrichment by e.g. phage display is needed prior to thescreening. However, in the screening of combinatorial libraries, theimmunoassays is preferably performed in combination with or followingenrichment methods such as phage display, ribosome display, bacterialsurface display, yeast display, eukaryotic virus display, RNA display orcovalent display (reviewed in FitzGerald, K., 2000. Drug Discov. Today5, 253-258).

The V_(H) and V_(L) pair encoding sequences selected in the screeningare generally subjected to sequencing, and analyzed with respect todiversity of the variable regions. In particular the diversity in theCDR regions is of interest, but also the V_(H) and V_(L) familyrepresentation is of interest. Based on these analyses, sequencesencoding V_(H) and V_(L) pairs representing the overall diversity of theorthopoxvirus binding antibodies isolated from one or more donors areselected. Preferably, sequences with differences in all the CDR regions(CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3) are selected. If thereare sequences with one or more identical or very similar CDR regionswhich belong to different V_(H) or V_(L) families, these are alsoselected. The selection of V_(H) and V_(L) sequence pairs can also beperformed based on the diversity of the CDR3 region of the variableheavy chain. During the priming and amplification of the sequences,mutations may occur in the framework regions of the variable region.Preferably, such errors are corrected in order to ensure that thesequences correspond completely to those of the donor, e.g. such thatthe sequences are completely human in all conserved regions such as theframework regions of the variable region.

When it is ensured that the overall diversity of the collection ofselected sequences encoding V_(H) and V_(L) pairs is highlyrepresentative of the diversity seen at the genetic level in a humeralresponse to an orthopoxvirus challenge, it is expected that the overallspecificity of antibodies expressed from a collection of selected V_(H)and V_(L) coding pairs, also are representative with respect to thespecificity of the antibodies produced in the challenged donors. Anindication of whether the specificity of the antibodies expressed from acollection of selected V_(H) and V_(L) coding pairs, are representativeof the specificity of the antibodies raised by challenged donors can beobtained by comparing the antibody titers towards the virus strains aswell as the selected antigens of the donor blood with the specificity ofthe antibodies expressed from a collection of selected V_(H) and V_(L)coding pairs. Additionally, the specificity of the antibodies expressedfrom a collection of selected V_(H) and V_(L) coding pairs can beanalyzed further. The degree of specificity correlates with the numberof different antigens towards which binding reactivity can be detected.In a further embodiment of the present invention the specificity of theindividual antibodies expressed from a collection of selected V_(H) andV_(L) coding pairs are analyzed by Western blot. Briefly, the antigensfrom an orthopoxvirus strain are resolved on polyacrylamide gel, underreducing conditions. The antibodies are analyzed individually in aWestern blot procedure, identifying the protein antigens to which theybind. The binding pattern of the individual antibodies is analyzed andcompared to the other antibodies expressed from a collection of selectedV_(H) and V_(L) coding pairs. Preferably, individual members to becomprised in an anti-orthopoxvirus rpAb of the present invention areselected such that the specificity of the antibody compositioncollectively covers all the antigens which have produced significantantibody titers in a serum sample from the donor(s). Even morepreferred, antibodies with different binding pattern in the Western blotanalysis are selected to constitute an anti-orthopoxvirus rpAb of thepresent invention.

Production of a Recombinant Polyclonal Antibody from Selected V_(H) andV_(L) Coding Pairs

A polyclonal antibody of the present invention is produced from apolyclonal expression cell line in one or a few bioreactors orequivalents thereof. Following this approach the anti-orthopoxvirus rpAbcan be purified from the reactor as a single preparation without havingto separate the individual members constituting the anti-orthopoxvirusrpAb during the process. If the polyclonal antibody is produced in morethan one bioreactor, the supernatants from each bioreactor can be pooledprior to the purification, or the purified anti-orthopoxvirus rpAb canbe obtained by pooling the antibodies obtained from individuallypurified supernatants from each bioreactor.

One way of producing a recombinant polyclonal antibody is described inWO 04/061104 and PCT/DK2005/000501 (these references are herebyincorporated by reference). The method described therein, is based onsite-specific integration of the antibody coding sequence into thegenome of the individual host cells, ensuring that the V_(H) and V_(L)protein chains are maintained in their original pairing duringproduction. Further, the site-specific integration minimizes positioneffects and therefore the growth and expression properties of theindividual cells in the polyclonal cell line are expected to be verysimilar. Generally, the method involves the following: i) a host cellwith one or more recombinase recognition sites; ii) an expression vectorwith at least one recombinase recognition site compatible with that ofthe host cell; iii) generation of a collection of expression vectors bytransferring the selected V_(H) and V_(L) coding pairs from thescreening vector to an expression vector such that a full-lengthantibody or antibody fragment can be expressed from the vector; iv)transfection of the host cell with the collection of expression vectorsand a vector coding for a recombinase capable of combining therecombinase recognition sites in the genome of the host cell with thatin the vector; v) obtaining/generating a polyclonal cell line from thetransfected host cell and vi) expressing and collecting the polyclonalantibody from the polyclonal cell line.

Preferably mammalian cells such as CHO cells, COS cells, BHK cells,myeloma cells (e.g., Sp2/0 or NSO cells), fibroblasts such as NIH 3T3,and immortalized human cells, such as HeLa cells, HEK 293 cells, orPER.C6, are used. However, non-mammalian eukaryotic or prokaryoticcells, such as plant cells, insect cells, yeast cells, fungi, E. colietc., can also be employed. A suitable host cell comprises one or moresuitable recombinase recognition sites in its genome. The host cellshould also contain a mode of selection which is operably linked to theintegration site, in order to be able to select for integrants, (i.e.,cells having an integrated copy of an anti-orthopoxvirus Ab expressionvector or expression vector fragment in the integration site). Thepreparation of cells having an FRT site at a pre-determined location inthe genome was described in e.g. U.S. Pat. No. 5,677,177. Preferably, ahost cell only has a single integration site, which is located at a siteallowing for high expression of the integrant (a hot-spot).

A suitable expression vector comprises a recombination recognition sitematching the recombinase recognition site(s) of the host cell.Preferably the recombinase recognition site is linked to a suitableselection gene different from the selection gene used for constructionof the host cell. Selection genes are well known in the art, and includeglutamine synthetase gene (GS) and neomycin. The vector may also containtwo different recombinase recognition sites to allow forrecombinase-mediated cassette exchange (RMCE) of the antibody codingsequence instead of complete integration of the vector. RMCE isdescribed in Langer et al 2002, Nucleic Acids Res. 30, 3067-3077;Schlake and Bode 1994, Biochemistry 33, 12746-12751 and Belteki et al2003, Nat. biotech. 21, 321-324. Suitable recombinase recognition sitesare well known in the art, and include FRT, lox and attP/attB sites.Preferably the integrating vector is an isotype-encoding vector, wherethe constant regions (preferably including introns) are present in thevector prior to transfer of the V_(H) and V_(L) coding pair from thescreening vector. The constant regions present in the vector can eitherbe the entire heavy chain constant region (CH₁ to CH₃ or to CH₄) or theconstant region encoding the Fc part of the antibody (CH₂ to CH₃ or toCH₄). The light chain Kappa or Lambda constant region may also bepresent prior to transfer. The choice of the number of constant regionspresent, if any, depends on the screening and transfer system used. Theheavy chain constant regions can be selected from the isotypes IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. Preferred isotypes areIgG1 and/or IgG3. Further, the expression vector for site-specificintegration of the anti-orthopoxvirus antibody coding nucleic acidcontains suitable promoters or equivalent sequences directing highlevels of expression of each of the V_(H) and V_(L) chains. FIG. 4illustrates one possible way to design the expression vector, althoughnumerous other designs are possible.

The transfer of the selected V_(H) and V_(L) coding pairs from thescreening vector can be performed by conventional restriction enzymecleavage and ligation, such that each expression vector molecule containone V_(H) and V_(L) coding pair. Preferably, the V_(H) and V_(L) codingpairs are transferred individually, they may, however, also betransferred in-mass if desired. When all the selected V_(H) and V_(L)coding pairs are transferred to the expression vector a collection or alibrary of expression vectors is obtained. Alternative ways of transfermay also be used if desired.

Methods for transfecting a nucleic acid sequence into a host cell areknown in the art. To ensure site-specific integration a suitablerecombinase must be provided to the host cell as well. This ispreferably assured by co-transfection of a plasmid encoding therecombinase. Suitable recombinases are for example Flp, Cre or phageΦC31 integrase, when used together with a host cell/vector system withthe corresponding recombinase recognition sites. The host cell caneither be transfected in bulk, meaning that the library of expressionvectors is transfected into the cell line in one single reaction therebyobtaining a polyclonal cell line. Alternatively, the collection ofexpression vectors can be transfected individually into the host cell,thereby generating a collection of individual cell lines (producingmonoclonal antibodies). The cell lines generated upon transfection(monoclonal or polyclonal) are then selected for site specificintegrants, and adapted to grow in suspension and serum free media, ifthey did not already do this prior to transfection. If the transfectionwas performed individually, the individual cell lines are analyzedfurther with respect to their grow properties and antibody production.Preferably cell lines with similar proliferation rates and antibodyexpression levels are selected for the generation of the polyclonal cellline. The polyclonal cell line is then generated by mixing theindividual cell lines in a predefined ratio. Generally, a polyclonalmaster cell bank (pMCB) and/or a polyclonal working cell bank (pWCB) islaid down from the polyclonal cell line.

One embodiment of the present invention is a polyclonal cell linecapable of expressing a recombinant polyclonal anti-orthopoxvirusantibody of the present invention.

A further embodiment of the present invention is a polyclonal cell linewherein each individual cell is capable of expressing a single V_(H) andV_(L) coding pair, and the polyclonal cell line as a whole is capable ofexpressing a collection of V_(H) and V_(L) coding pairs, where eachV_(H) and V_(L) coding pair encode an anti-orthopoxvirus antibody of thepresent invention. Preferably the collection of V_(H) and V_(L) codingpairs are cognate pairs generated according to the methods of thepresent invention.

The recombinant polyclonal antibody is then expressed by culturing oneampoule from the pWCB in an appropriate medium for a period of timeallowing for sufficient expression of antibody and where the polyclonalcell line remains stable (The window is approximately between 15 daysand 50 days). Culturing methods such as fed batch or perfusion may beused. The recombinant polyclonal antibody is obtained from the culturemedium and purified by conventional purification techniques. Affinitychromatography combined with subsequent purification steps such asion-exchange chromatography, hydrophobic interactions and gel filtrationhas frequently been used for the purification of IgG. Followingpurification, the presence of all the individual members in thepolyclonal antibody composition is assessed, for example by ion-exchangechromatography. The characterization of a polyclonal antibodycomposition is described in detail in PCT/DK2005/000504 (herebyincorporated by reference).

An alternatively method of expressing a mixture of antibodies in arecombinant host is described in WO 04/009618, this method producesantibodies with different heavy chains associated with the same lightchain from a single cell line. This approach may be applicable if theanti-orthopoxvirus rpAb is produced from a combinatorial library.

Therapeutic Compositions

Another aspect of the invention is a pharmaceutical compositioncomprising as an active ingredient anti-orthopoxvirus rpAb oranti-orthopoxvirus recombinant polyclonal Fab or anotheranti-orthopoxvirus recombinant polyclonal fragment. Preferably, theactive ingredient of such a composition is an anti-orthopoxvirusrecombinant polyclonal antibody as claimed by the present invention.Such compositions are intended for prevention, and treatment of adverseeffects of vaccination with vaccinia virus or treatment of orthopoxvirusinfections, in particular infections with variola virus ormonkeypoxvirus. Preferably, the treatment is administered to a human adomestic animal or a pet.

The pharmaceutical composition further comprises a pharmaceuticallyacceptable excipient.

In further embodiments of the present invention, any of the previouslydescribed anti-orthopoxvirus Ab compositions (polyclonal or monoclonal)may additionally be combined with other compositions for the treatmentof an orthopoxvirus infection, such as Cidofovir, STI-571 (Reeves et al.2005, Nature Med. 11:731-739) and/or ST-246 (Yang et al. 2005, J. Virol.79:13139-13149).

Anti-orthopoxvirus rpAb or polyclonal fragments thereof may beadministered within a pharmaceutically-acceptable diluent, carrier, orexcipient, in unit dose form. Conventional pharmaceutical practice maybe employed to provide suitable formulations or compositions toadminister to individuals being vaccinated with an orthopoxvirus orpatients showing adverse effects following vaccination or patientsinfected with an orthopoxvirus. In a preferred embodiment theadministration is prophylactic. Any appropriate route of administrationmay be employed, for example, administration may be parenteral,intravenous, intra-arterial, subcutaneous, intramuscular,intraperitoneal, intranasal, aerosol, suppository, or oraladministration. For example, therapeutic formulations may be in the formof, liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules chewing gum orpasta, and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example, by means of conventional dissolving,lyophilizing, mixing, granulating or confectioning processes. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see for example, in Remington: The Science andPractice of Pharmacy (20th ed.), ed. A. R. Gennaro, 2000, LippincottWilliams & Wilkins, Philadelphia, Pa. and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York, N.Y.).

Solutions of the active ingredient, and also suspensions, and especiallyisotonic aqueous solutions or suspensions, are preferably used, it beingpossible, for example in the case of lyophilized compositions thatcomprise the active ingredient alone or together with a carrier, forexample mannitol, for such solutions or suspensions to be produced priorto use. The pharmaceutical compositions may be sterilized and/or maycomprise excipients, for example preservatives, stabilizers, wettingand/or emulsifying agents, solubilizers, salts for regulating theosmotic pressure and/or buffers, and are prepared in a manner known perse, for example by means of conventional dissolving or lyophilizingprocesses. The said solutions or suspensions may compriseviscosity-increasing substances, such as sodium carboxymethylcellulose,carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

The injection compositions are prepared in customary manner understerile conditions; the same applies also to introducing thecompositions into ampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration can be obtained bycombining the active ingredient with solid carriers, if desiredgranulating a resulting mixture, and processing the mixture, if desiredor necessary, after the addition of appropriate excipients, intotablets, pills, or capsules, which may be coated with shellac, sugar orboth. It is also possible for them to be incorporated into plasticscarriers that allow the active ingredients to diffuse or be released inmeasured amounts.

The pharmaceutical compositions comprise from approximately 1% toapproximately 95%, preferably from approximately 20% to approximately90%, active ingredient. Pharmaceutical compositions according to theinvention may be, for example, in unit dose form, such as in the form ofampoules, vials, suppositories, tablets, pills, or capsules. Theformulations can be administered to human individuals in therapeuticallyor prophylactic effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for adisease or condition. The preferred dosage of therapeutic agent to beadministered is likely to depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

Therapeutic Uses of the Compositions According to the Invention

The pharmaceutical compositions according to the present invention maybe used for the treatment, amelioration or prophylaxis of a disease in amammal. Conditions that can be treated or prevented with the presentpharmaceutical compositions include prevention, and treatment of adverseeffects of vaccination with vaccinia virus or other orthopoxvirus andtreatment of individuals infected with an orthopoxvirus, in particularvariola virus, monkeypox virus and camelpox virus infections.

One embodiment of the present invention is a method for treatment orprophylaxis of an orthopoxvirus infection in a human or animal, whereinan effective amount of an anti-orthopoxvirus recombinant polyclonal ofthe present invention is administered to said human or animal.

A further embodiment of the present invention is a method for treatment,or prevention of adverse side effects of vaccination with vaccinia virusin a human or an animal, wherein an effective amount of ananti-orthopoxvirus recombinant polyclonal of the present inventionadministered to said human or animal.

A further embodiment of the present invention is the use of ananti-orthopoxvirus recombinant polyclonal antibody of the presentinvention for the preparation of a composition for the treatment, orprevention of adverse side effects of vaccination with vaccinia virus,or for treatment or prophylaxis of orthopoxvirus infections.

Diagnostic Use and Environmental Detection Use

Another embodiment of the invention is directed to diagnostic kits. Kitsaccording to the present invention comprise an anti-orthopoxvirus rpAbprepared according to the invention which protein may be labeled with adetectable label or non-labeled for non-label detection. The kit may beused to identify individuals infected with orthopoxvirus.

EXAMPLES Example 1

This example is a collection of the methods applied to illustrate thepresent invention.

a. Sorting of Plasma Cells from Donor Blood

The peripheral blood mononuclear cells (PBMC) were isolated from blooddrawn from donors using Lymphoprep (Axis Shield) and gradientcentrifugation according to the manufactures instructions. The isolatedPBMC were either cryopreserved in FCS; 10% DMSO at −150° C. or useddirectly. The B cell fraction was labeled with anti-CD19 antibody andisolated from the PBMC fraction using magnetic cell sorting (MACS). ThePBMC (1×10⁶ cells) were incubated with anti-CD19-FITC conjugatedantibody (BD Pharmingen) for 20 minutes at 4° C. Cells were washed twicein, and resuspended in MACS buffer (Miltenyi Biotec). Anti-FITCMicroBeads (Miltenyi Biotec) were mixed with the labeled cells andincubated for 15 minutes at 4° C. The washing procedure was repeatedbefore the cell-bead suspension was applied to a LS MACS column(Miltenyi Biotec). The CD19 positive cell fraction was eluted from thecolumn according to the manufactures instructions and either stored inFCS-10% DMSO, or proceeded directly to single cell sorting.

Plasma blasts or circulating plasma cells (hereafter plasma cells) wereselected from the CD19⁺ B cell fraction by fluorescence activated cellsorting (FACS) based on the expression profile of CD19, CD38, and CD45cell surface proteins. CD19 is a B-cell market that is also expressed onearly plasma cells, while CD38 is highly expressed on plasma cells. Theplasma cells apparently, have a somewhat lower expression of CD45 thanthe rest of the CD19⁺ cells, which allow separation of a discretepopulation. The MACS purified cells were thawed or used directly. Thecells were washed in FACS buffer (PBS; 1% BSA) and stained for 20 minutewith CD19-FITC, CD38-PE, CD45-PerCP (BD Pharmingen). The cells werewashed and re-suspended in FACS buffer.

The flow rate of the cells during the FACS was set at 200 events/sec andthe cell concentration was 5×10⁵/ml to obtain a high plasma cellsrescue. The following set of gates, were used. Each gate is a daughterof the former.

-   Gate 1: FSC/SSC gate. The lymphocyte population having the highest    FSC is selected ensuring sorting of living cells.-   Gate 2: SSCh/SSCw. This gate ensures sorting of single cells.-   Gate 3: CD19⁺ cells. In the FL1-FL2 dot plot, only the CD19 positive    cells are selected.-   Gate 4: In the FL2-FL3 dot plot, a discrete population should be    visible, which is CD38high and CD45intermediate.

The resulting population that fulfills these four criteria wassingle-cell sorted into 96-well PCR plates containing a sorting buffer(see section c). The cell containing plates were stored at −80° C.

b. ELISpot

ELISpot was used to estimate the percentages of plasma cells expressinganti-vaccinia virus antibodies in obtained cell samples i.e. PBMC, MACSpurified CD19+ cells, or a population of FACS sorted plasma cells.96-well plates with a nitrocellulose surface (Millipore) were coatedwith a solution of 20 μg/ml inactivated vaccinia virus particles ofeither Lister strain of IHD-W strain (AutogenBioclear, UK). The wellswere blocked by incubation with RPMI, 10% FCS at 4° C. over night. Theplasma cell containing cell sample was added in RPMI culture medium toeach well followed by incubation at standard tissue culture conditionsfor 24 hours. The secreted vaccinia virus specific antibodies will bindto the immobilized virus particles surrounding the antibody producingplasma cell. The cells were removed by washing three times in PBS; 0.01%Tween20 and three times in PBS. HRP-conjugated anti-human IgG (H+L)(CalTag) and HRP-conjugated anti-human IgA (SeroTech) were added andallowed to react with the immobilized antibodies for 1 hour at 37° C.The washing procedure was repeated and the chromogen substrate(3-amino-9-ethylcarbazole solubilized in N,N-DMF (di-methyl formamide)was added. The color development was terminated after 4 minutes byaddition of H₂O. Red spots were identified at the sites where antigenspecific plasma cells had been located.

c. Linkage of Cognate V_(H) and V_(L) Pairs

The linkage of V_(H) and V_(L) coding sequences was performed on thesingle cells obtained as described in section a), facilitating cognatepairing of the V_(H) and V_(L) coding sequences. The procedure was a twostep PCR procedure based on a one-step multiplex overlap-extensionRT-PCR followed by a nested PCR. The primer mixes used in the presentexample only amplify Kappa light chains. Primers capable of amplifyingLambda light chains could, however, be added to the multiplex primer mixand nested PCR primer mix if desired. The principle is illustrated inFIG. 2.

The 96-well PCR plates from produced in step a) were thawed. The singlecell served as template for the multiplex overlap-extension RT-PCR.Sorting buffer containing reaction buffer (Phusion HF buffer;Finnzymes), primers for RT-PCR (see Table 2) and RNase inhibitor(RNasin, Promega) was already added to each well before the single-cellsorting. The following was added to each well to obtain the given finalconcentration: dNTP mix (200 μM each), RNAse inhibitor (20 U/μl),Sensiscript Reverse Transcriptase (320× dilution; Qiagen) and PhusionDNA Polymerase (0.4 U; Finnzymes).

The plates were incubated for 1 hour at 37° C. to allow reversetranscription of the RNA from each cell. Following the RT, the plateswere subjected to the following PCR cycle: 30 sec. at 98° C., 30× (20sec. at 98° C., 30 sec. at 60° C., 45 sec. at 72° C.), 45 sec. at 72° C.

The PCR reactions were performed in H20BIT Thermal cycler with Peel SealBasket for 24×96-well plates (ABgene), to facilitate a high-throughput.The PCR plates were stored at −20° C. after cycling.

TABLE 2 RT-PCR multiplex overlap-extension primer mix Final Primer Conc.name μM Sequence SEQ ID VH set CH-IgG 0.2 GACSGATGGGCCCTTGGTGG  1 CH-IgA0.2 GAGTGGCTCCTGGGGGAAGA  2 VH-1 0.04TATTCCCATGGCGCGCCCAGRTGCAGCTGGTGCART  3 VH-2 0.04TATTCCCATGGCGCGCCSAGGTCCAGCTGGTRCAGT  4 VH-3 0.04TATTCCCATGGCGCGCCCAGRTCACCTTGAAGGAGT  5 VH-4 0.04TATTCCCATGGCGCGCCSAGGTGCAGCTGGTGGAG  6 VH-5 0.04TATTCCCATGGCGCGCCCAGGTGCAGCTACAGCAGT  7 VH-6 0.04TATTCCCATGGCGCGCCCAGSTGCAGCTGCAGGAGT  8 VH-7 0.04TATTCCCATGGCGCGCCGARGTGCAGCTGGTGCAGT  9 VH-8 0.04TATTCCCATGGCGCGCCCAGGTACAGCTGCAGCAGTC 10 LC set CK1 0.2ATATATATGCGGCCGCTTATTAACACTCTCCCCTGTTG 11 VL-1 0.04GGCGCGCCATGGGAATAGCTAGCCGACATCCAGWTGA 12 CCCAGTCT VL-2 0.04GGCGCGCCATGGGAATAGCTAGCCGATGTTGTGATGA 13 CTCAGTCT VL-3 0.04GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGWTGA 14 CRCAGTCT VL-4 0.04GGCGCGCCATGGGAATAGCTAGCCGATATTGTGATGA 15 CCCACACT VL-5 0.04GGCGCGCCATGGGAATAGCTAGCCGAAACGACACTCA 16 CGCAGT VL-6 0.04GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGCTGA 17 CTCAGTCT W = A/T, R = A/G, S =G/C

For the nested PCR step, 96-well PCR plates were prepared with thefollowing mixture in each well to obtain the given final concentration:1× FastStart buffer (Roche), dNTP mix (200 μM each), nested primer mix(see Table 3), Phusion DNA Polymerase (0.08 U, Finnzymes) and FastStartHigh Fidelity Enzyme blend (0.8 U; Roche). As template for the nestedPCR, 1 μl was transferred from the multiplex overlap-extension PCRreactions. The nested PCR plates were subjected to the following PCRcycle: 35× (30 sec. at 95° C., 30 sec. at 60° C., 90 sec. at 72° C.),600 sec. at 72° C.

Selected reactions were analyzed on a 1% agarose gel to verify thepresence of an overlap-extension fragment of approximately 1070 bp.

The plates were stored at −20° C. until further processing of the PCRfragments.

TABLE 3 Nested primer set Final Primer Conc. SEQ name μM Sequence ID CK20.2 ACCGCCTCCACCGGCGGCCGCTTATTAACACTCTCCCCT 18 GTTGAAGCTCTT PJ 1-2 0.2GGAGGCGCTCGAGACGGTGACCAGGGTGCC 19 PJ 3 0.2GGAGGCGCTCGAGACGGTGACCATTGTCCC 20 PJ 4-5 0.2GGAGGCGCTCGAGACGGTGACCAGGGTTCC 21 PJ 6 0.2GGAGGCGCTCGAGACGGTGACCGTGGTCCC 22d. Insertion of Cognate V_(H) and V_(L) Coding Pairs Into a ScreeningVector

In order to identify antibodies with binding specificity to vacciniavirus particles, the V_(H) and V_(L) coding sequences were expressedeither as Fabs or full-length antibodies. This involved insertion of therepertoire of V_(H) and V_(L) coding pairs into a screening vector andtransformation into a host cell.

A two-step cloning procedure was employed for generation of a repertoireof screening vectors containing the V_(H) and V_(L) coding pairs.Statistically, if the repertoire of screening vectors contain ten timesas many recombinant plasmids as the number of cognate paired V_(H) andV_(L) PCR products used for generation of the screening repertoire,there is 99% likelihood that all unique gene pairs are represented.Thus, if 400 overlap-extension V-gene fragments were obtained in sectionc) a repertoire of at least 4000 clones was generated for screening.

Briefly, the repertoire of linked V_(H) and V_(L) coding pairs from thenested PCR in section c) were pooled (without mixing pairs fromdifferent donors). The PCR fragments were cleaved with XhoI and NotI DNAendonucleases at the recognition sites introduced into the termini ofPCR products. The cleaved and purified fragments were ligated into anXhoI/NotI digested Fab-expression vector by standard ligationprocedures. Suitable vectors were for example the bacterial or mammalianexpression vectors illustrated in FIG. 3. The ligation mix waselectroporated into E. coli and added to 2xYT plates containing theappropriated antibiotic and incubated at 37° C. over night. Theamplified repertoire of vectors was purified from cells recovered fromthe plates using standard DNA purification methods (Qiagen). Theplasmids were prepared for insertion of promoter-leader fragments bycleavage using AscI and NheI endonucleases. The restriction sites forthese enzymes were located between the V_(H) and V_(L) coding genepairs. Following purification of the vector, an AscI-NheI digestedbi-directional promoter-leader fragment was inserted into the AscI andNheI restriction sites by standard ligation procedures. The ligatedvector was amplified in E. coli and the plasmid was purified usingstandard methods. Where a bacterial screening vector was used thebi-directional promoters were bacterial promoters, and where themammalian screening vector was used the bi-directional promoters weremammalian promoters. The generated repertoire of screening vectors wastransformed into E. coli for by conventional procedures. Coloniesobtained were consolidated into 384-well master plates and stored. Thenumber of arrayed colonies exceeded the number of input PCR products byat least 3 fold, thus giving 95% percent likelihood for presence of allunique V-gene pairs obtained in section c).

e. Screening

The screening strategy is presented in FIG. 6. Fab expression wasperformed by inoculation of the colonies from the master plate into a384-well plate. In cases where Fab expression was performed from E. colithe plates contained 0.9 ml 2xYT, 0.1% glucose, 50 μg/ml Carbencilin andthe colonies were incubated with vigorous shaking at 37° C. until thecell density detected as OD600 reached ˜1. The Fab expression wasinduced by addition of 0.1 ml 2xYT, 0.1 M IPTG, 50 μg/ml Carbenicillinand the temperature decreased to 30° C. The Fab-containing supernatantswere cleared by centrifugation and stored for screening reactions. TheFab-containing supernatants were cleared by centrifugation and storedfor screening reactions. In cases where Fab expression was performedfrom mammalian vectors, DNA for transfection was prepared from the E.coli master plates. CHO cells were seeded into 384-well cell cultureplates (3000 cells per well) in F12-HAM medium with 10% fetal calf serum(FCS) and after an overnight incubation the cells were transfected withthe DNA using Fugene 6 as transfection agent. After 2-3 days in cellculture the Fab-containing supernatants were harvested and stored forscreening reactions.

Screening of individual clones was performed using a fluorescence-linkedimmunosorbent assay (FLISA) based on the fluorometric microvolume assaytechnology (FMAT) (Swartzman et al. 1999, Anal. Biochem. 271:143-151).Briefly, inactivated virus particles of the Lister strain, IHD-W strain,and the recombinant protein antigens B5R, VCP and A33R were immobilizedindividually on polystyrene beads (6.79 μm diameter, Spherotech Inc.) byincubating 16.5 μg protein or 20 μg virus particles with 100 μL 5% w/vbeads. The supernatant containing Fab-fragments were screened againstall five populations of coated beads. The coated beads, a secondaryfluorescently-labeled anti-human antibody (Alexa Dye 647, MolecularProbes) and supernatant containing Fab-fragments were mixed in 384-wellplates. Wells containing Fabs with reactivity against the coated antigenresulted in an increased fluorescence at the bead surface, which weredetected using FLISA reader (8200 Cellular Detection System; AppliedBiosystems). In principle, the assay is equivalent to ELISA but since nowashing steps are included, the procedure has a high throughput. Cut-offwas set at as low as 50 detected fluorescent beads (counts) during theprimary screen in order to minimize losses and to identify as manyclones reactive with viral particles or antigen as possible. From theoriginal master plates, the primary hits were retrieved and collected inwells of 96-well plates. The generated primary hit plates were handledas master plates, including storage and re-expression of these primaryhits. These re-expressed Fab molecules were tested in a secondaryscreening using the same antigens in both FLISA and standard ELISA.Clones which produced Fabs with reactivity in both FLISA and ELISA inthe secondary screen were submitted for DNA sequencing of the V-generegion.

The obtained sequences were aligned based on the amino acid sequence ofthe CDR3 region and grouped into clusters of clones expressing identicalFabs, some of the clusters only contained a single clone (a singleton).Large scale batches of Fab-fragments of representative clones from eachcluster were prepared for validation of the anti-vaccinia virusreactivity. These studies consisted of binding analyses by ELISA usinginactivated IHD-W, inactivated IHD-J, inactivated Lister strain VVparticles, and the recombinant antigens, A27L, L1R, B5R, VCP, and A33R.Clones producing Fab-fragments which were positive to an inactivatedvaccinia virus strain and/or one of the recombinant antigens were termedvalidated

f. Transfer of Selected Clones to Mammalian Expression Vector

When using a multiplex PCR approach as described in section c), acertain degree of intra-V-gene family cross-priming and inter-V-genefamily cross-priming is expected due to the high degree of homology. Thecross-priming introduces non-natural occurring amino acids into thesequences with several potential consequences e.g. structural changesand increased immunogenicity, all resulting in a decreased therapeuticactivity.

In order to eliminate these drawbacks and to ensure that selected clonesmirror the natural humeral immune response such cross-priming mutationswere corrected during the transfer of the V_(H) and V_(L) coding pairs(in the form of the complete light chain linked to the variable heavychain) from the screening vector to the mammalian expression vector.Either all clones, mutations or not, or just the clones with mutationswere subjected to the two-step transfer procedure.

In the first step of the repair transfer procedure, the V_(H) sequencewas PCR amplified with a primer set containing the sequencecorresponding to the originating V_(H)-gene family thereby reversemutating any cross-priming introduced changes. The PCR fragment wasdigested with XhoI and AscI and ligated into the XhoI/AscI digestedmammalian expression vector (FIG. 3) using conventional ligationprocedures. The ligated vector was amplified in E. coli and the plasmidwas purified using standard methods. The V_(H) sequence was sequenced toverify the correction. The vector was digested with NheI/NotI, toprepare it for insertion of the light chain.

In the second step the complete light chain was PCR amplified with aprimer set containing the sequence corresponding to the originatingV_(L)-gene thereby reverse mutating any cross-priming changes. The PCRfragment was digested with NheI/NotI and ligated into the V_(H)containing vector prepared above. The ligation product was amplified inE. coli and the plasmid was purified. The light chain was sequenced toverify the correction.

If clones did not need correction of the V_(H) and V_(L) coding pair,they could optionally be transferred directly as a pair in a single stepusing the XhoI/NheI restriction sites in a conventional cloningprocedure. In that event a promoter change was necessary if thescreening vector was a bacterial vector. This was performed as describedin section d). If the transfer was performed from a mammalian screeningvector no promoter exchange was necessary.

g. Generation of a Polyclonal Cell Line

The generation of a polyclonal expression cell line producing arecombinant polyclonal antibody is a multi-step procedure involving thegeneration of individual expression cell lines (monoclonal cell lines)which each express a unique antibody. The polyclonal cell line isobtained by mixing the individual cell lines thereby generating apolyclonal master cell bank (pMCB) from which a polyclonal working cellbank (pWCB) can be generated simply by continuing amplification.

h. Transfection and Selection of Mammalian Cell Lines

The Flp-In CHO cell line (Invitrogen) was used as starting cell line. Inorder to obtain a more homogenous cell line the parental Flp-In CHO cellline was sub-cloned by limited dilution and several clones were selectedand expanded. Based on growth behavior one clone, CHO-Flp-In (019), wasselected as starting cell line. The CHO-Flp-In (019) cells were culturedas adherent cells in F12-HAM with 10% fetal calf serum (FCS).

The individual plasmid preparations each containing a selected V_(H) andV_(L) coding pair obtained in step 0), were co-transfected with Flprecombinase encoding plasmid into 5×10⁶ CHO-Flp-In (019) cell line usingFugene6 (Roche) (for further details see WO 04/061104) to generateapproximately 10,000 independent recombination events for eachtransformation. The large-scale transformation procedure was applied asuniformly as possibly to ensure that identical expression cellpopulations were generated for each V_(H) and V_(L) coding pair. Cellswere trypsinated after 24 hours and transferred to 3×T175 flasks.Recombinant cell lines were selected by culturing in the presence of 450μg/ml Neomycin, which was added 48 hours after transfection.Approximately two weeks later clones appeared. Clones were counted andcells were trypsinated and hereafter cultured as pools of clonesexpressing one of the specific anti-VV antibodies.

i. Adaptation to Serum Free Suspension Culture

The individual adherent anti-VV antibody CHO-Flp-In (019) cell cultureswere trypsinated, centrifuged and transferred to separate shaker flaskswith 8×10⁵ cells/ml in appropriate serum free medium (Excell 302, JRHBiosciences). Growth and cell morphology were followed over severalweeks. After 4-6 weeks the cell lines usually showed good and stablegrowth behavior with doubling times below 32 hours and the adaptedindividual cell line was cryopreserved as described above.

j. Characterization of Cell Lines

All the individual cell lines were characterized with respect toantibody production and proliferation. This was performed with thefollowing assays:

Production:

The production of recombinant antibodies of the individual expressioncell lines were followed during the adaptation by Kappa specific ELISA.ELISA plates were coated overnight with goat-anti-human Kappa antibodies(Caltag) in carbonate buffer, pH 9.6. Plates were washed 6 times withwashing buffer (PBS; 0.05% Tween 20) and blocked by incubation for 1hour in washing buffer containing 2% non fat milk. Cell culture mediasupernatants were added and the incubated extended for 1 hour. Plateswere washed 6 times in washing buffer and secondary antibodies(goat-anti-human IgG (H+L) HRPO, Caltag) were added and the incubationrepeated. After vigorous washing the ELISA was developed with TMBsubstrate and reaction stopped by addition of H₂SO₄. Plates were read at450 nm.

Further, intracellular staining was used to determine the generalexpression level as well as to determine the homogeneity of the cellpopulation in relation to expression of recombinant antibody. 5×10⁵cells were washed in cold FACS buffer (PBS; 2% FCS) before fixation byincubation in CellFix (BD-Biosciences) for 20 minutes and hereafterwashed in saponin buffer (PBS; 0.2% Saponin). The suspension wascentrifuged and fluorescently tagged antibody (Goat F(ab′)₂ Fragment,Anti-human IgG(H+L)-PE, Beckman Coulter) was added. After 20 minutes onice the cells were washed twice in saponin buffer and suspended in FACSbuffer and analyzed by FACS.

Proliferation:

Aliquots of the cell suspensions were taken three times a week and cellnumber, cell size, degree of clumping, and viability was determined byCASY® (Cell Counter+Analyzer System from Schärfe System GmbH) analysis.The doubling time for the cell cultures was calculated by cell numberderived form CASY® measurements.

k. Characterization of the Antigen Specificity of the IndividualAntibodies

The antigen specificity of the individual expressed antibodies wasassessed in order to allow the generation of an anti-VV rpAb with awell-characterized specificity. As already described in section e) theantibodies identified during screening were validated by assessing theirbinding specificity to inactivated vaccinia virus strains andrecombinantly produced A27L, L1R, B5R, VCP, and A33R antigens. Theseanalyses were repeated using the full-length antibodies and additionalanalysis of the antigen specificity was performed by Western blotting.

The vaccinia virus particle associated proteins (antigens) wereseparated by SDS-PAGE, using acetone precipitated virus particles whichsubsequently were dissolved in SDS-loading buffer containing 8 M ureaand run on a NuPAGE Bis-Tris 4-12% gel or NuPAGE Bis-Tris 10%. Thisresulted in a clear separation of the vaccinia virus particle associatedproteins when visualized by Coomassie blue. For antigen bindinganalysis, the vaccinia virus particle associated proteins were separatedby SDS-PAGE and electroblotted onto a PVDF membrane and purifiedpreparations of the individual antibodies were analyzed by Westernblotting. FIG. 5 shows Western blot analyses identifying a series ofantigens derived from the Lister strain detected by differentantibodies.

Putative target proteins were expressed by in vitro translation andtested for interaction with the selected antibodies by ELISA. Theantigen genes were generated by a gene specific PCR using vacciniavirus, lister strain DNA as template (Autogenbioclear, UK Cat. No.:08-940-250) followed by a second PCR step for addition of T7 promoterand Poly-A sequences. The Phusion DNA polymerase (Finnzymes, F cat. No:F530L) gene specific PCR reactions consisted of total volume at 50 μlcontaining 0.2 μM of 5′ and 3′ gene specific oligo pairs as indicated inTable 4, 1× Phusion HF reaction, 0.2 μM of each dNTP and 0.5 μl vacciniavirus DNA, 1 U Phusion DNA polymerase and were cycled through, 1×98° C.30 seconds, 25×(98° C. for 10 seconds, 50° C. for 15 seconds 72° C. 20seconds), 1×72° C. 7 minutes. The T7-Kozak translational initiation andPoly-A sequence were added by a second PCR reaction identical to theabove described except for that T7-Koz. Poly-A (Table 4) primers and 0.5μl of the 1 step PCR reaction were used as template. The PCR productswere in vitro translated using the TNT T7 Quick for PCR DNA kit(Promega, USA, Cat. No.: L1170) according to manufacturers procedure. Indetail each reaction consists of 40 μl TNT T7 Quick Master Mix, 1 μl mMMethionine 1, 1 μl Transcend Biotin-Lysyl-tRNA (Promega, Cat. No.L5061), 2 μl PCR generated DNA and incubated for 75 minutes at 30° C.

The in vitro translated antigens were tested for interactions withpurified antibodies by ELISA performed in standard ELISA. The purifiedantibodies were immobilized in an ELISA plate and incubated withdilutions of the in vitro translated antigens. Retained antigen wasdetected by Streptavidin Peroxidase Polymer (Sigma Cat. No. S-2438)according to standard ELISA procedure similar to the description inExample 1, section g).

TABLE 4 Primers for generation of in vitro translational  antigen genesPrimer SEQ Name Sequence ID H3L-5′GGGAACAGCCACCATGGCGGCGGTGAAAACTCCTGTTA 23 H3L-3′GGATCCTCTAGATCATTAAAATGAAATCAGTGGAGTAGTAAAC 24 A56R-5′GGGAACAGCCACCATGACATGACACCTTTTCCTCAGACATCT 25 A56R-3′GGATCCTCTAGATCATTAAGAGGTTGTACTACTACCTAC 26 B5R-5′GGGAACAGCCACCATGACATGTACTGTACCCACTATGA 27 B5R-3′GGATCCTCTAGATCATTATAACGATTCTATTTCTTG 28 Poly-ATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGATCCTCTAGATCATTA 29 T7-Koz.GGATCCTAATACGACTCACTATAGGGAACAGCCACCATG 30 A10L-5′GGGAACAGCCACCATGATGCCTATTAAGTCAATAG 31 A10L-3GGATCCTCTAGATCATTATTCATCATCAAAAGAG 32 A3L-5′GGGAACAGCCACCATGGAAGCCGTGGTCAATAGCGA 33 A3L-3′GGATCCTCTAGATCATTAAAATAGTTCTGTAATATGTCTA 34 A13L-5′GGGAACAGCCACCATGATTGGTATTCTTTTGTTGAT 35 A13L-3′GGATCCTCTAGATCATTATACAGAAGATTTAACTAGAT 36 D8L-5′GGGAACAGCCACCATGCCGCAACAACTATCTCCT 37 D8L-3′GGATCCTCTAGATCATTATTTATTCCCTTCGATATATTTTTGA 38 A11L-5′GGGAACAGCCACCATGACGACCGTACCAGTGACG 39 A11L-3′GGATCCTCTAGATCATTAAATAATTTTAATTCGTTTAA 40 A17L-5′GGGAACAGCCACCATGAGTTATTTAAGATATTACAAT 41 A17L-3′GGATCCTCTAGATCATTAATAATCGTCAGTATTTAAACT 42 L5R-5′GGGAACAGCCACCATGGAGAATGTTCCTAATGTA 43 L5R-3′GGATCCTCTAGATTATCATCTGCGAAGAACATCGTTA 44 F13L-5′GGGAACAGCCACCATGTGGCCATTTGCATCGGTA 45 F13L-3′GGATCCTCTAGATCATTAAATTTTTAACGATTTACTGT 46 A16L-5′GGGAACAGCCACCATGATGGGGGCAGCTGTTACTCTT 47 A16L-3′GGATCCTCTAGATCATTAAGGCAGTTTTATTTTATCTTTTA 48l. Characterization of the Biochemical Properties the IndividualAntibodies

Heterogeneity is a common phenomenon in antibodies and recombinantproteins.

Antibody modifications can occur during expression, through unfavorablestorage conditions, and may cause size or charge heterogeneity. Commonmodifications include N-glycosylation, methionine oxidation, proteolyticfragmentation, and deamidation. Since these parameters need to bewell-defined for therapeutic antibodies, they are analyzed prior to thegeneration of the polyclonal cell line.

The antibodies expressed during adaptation were purified by affinitychromatography (Protein-A columns) with low pH elution, and used forcharacterization of the biochemical properties of each individualantibody. The methods used for characterization included reducing andnon-reducing SDS-PAGE and weak cation exchange chromatography (IEX).Well-defined heavy and light chain bands in the reducing SDS-PAGEindicated intact antibodies. SDS-PAGE analysis of antibody preparationsresulting in well-defined bands, were expected to exert a single peakbehavior in IEX analysis, indicating a homogeneously antibodypreparation. Antibody preparations resulting in multiple peaks in theIEX analysis and/or aberrant migration of either the light or heavychain in SDS gels were analyzed in detail for intact N-termini byN-terminal sequencing, as well as for the presence of additionalN-glycosylation sites in the variable chains using enzymatic treatment.

m. Establishment of a Polyclonal Cell Line for Anti-VV RecombinantPolyclonal Antibody Production

Out of the pool of established expression cell lines a subset wereselected to constitute the polyclonal expression cell line (pMCB). Theselection parameters can be defined according to the use of thepolyclonal antibody to be produced from the polyclonal cell line and theperformance of the individual cell lines. Generally the followingparameters were considered:

-   -   Cell line characteristics; to optimize the stability of the        polyclonal cell line, individual cell lines with doubling times        between 21 and 34 hours and antibody productivity above 1        μg/cell/day.    -   Antigen reactivity; which antigens should the anti-VV rpAb exert        reactivity against, IMV, EEV and/or RCA derived proteins?    -   Protein chemistry; generally antibodies with well-defined        biochemical characteristics were included in the final anti-VV        rpAb.

The selected individual cell lines each expressing a recombinant anti-VVantibody were thawed and expanded at 37° C. in serum free medium inshaker flasks to reach at least 4×10⁸ cells of each clone having apopulation doubling time of 21-34 hours. The viabilities were in therange of 93% to 96%. The polyclonal cell line was prepared by mixing2×10⁶ cells from each cell line. The polyclonal cell line wasdistributed into freeze ampoules containing 5.6×10⁷ cells andcryopreserved. This collection of vials with a polyclonal cell line istermed the polyclonal master cell bank (pMCB) from which the polyclonalworking cell bank (pWCB) was generated by expanding one ampoule from thepMCB to reach a sufficient number of cells to lay down a polyclonalworking cell bank (pWCB) of approximately 200 ampoules with the samecell density as the ampoules of the pMCB. Samples in the cell banks weretested for mycoplasma and sterility.

n. Expression of a Recombinant Polyclonal Anti-VV Antibody

Recombinant polyclonal anti-VV antibody batches were produced in 5 literbioreactors (B. Braun Biotech International, Melsungen, Germany).Briefly, vials from the pWCB were thawed and expanded in shaker flasks(Coming). Cells in seed train were cultured in ExCell 302 medium withG418 and with anti-clumping agent at 37° C., 5% CO₂. The bioreactorswere inoculated with 0.6×10⁶ cells/ml suspended in 3 liter ExCell 302medium without G418 and without anti-clumping agent. The cellnumbers/viable cells were daily monitored by CASY counting. At 50 hours2000 ml ExCell 302 medium was supplemented and after 92 hours atemperature downshift from 37° C. to 32° C. was performed. The cellculture supernatant was harvested after 164 hours and subjected topurification as described in section o).

o. Purification of Anti-VV rpAb

The antibody expressed as described in section n) was of the IgG1isotype and affinity purified (Protein-A). The individual antibodiesinteracted with immobilized Protein A at pH 7.4, whereas contaminatingproteins were washed from the column. The bound antibodies weresubsequently eluted from the column by lowering of the pH to 2.7. Thefractions containing antibodies, determined from absorbance measurementsat 280 nm, were pooled and dialyzed against 5 mM sodium acetate, 150 mMNaCl, pH 5 and long time stored at -20° C.

p. In Vitro Neutralization Assays

Preparation of Vaccinia Virus for Use in Vivo and In Vitro

Lister, NYCBOH, and IHD-J vaccinia virus at 10⁴ pfu/ml was added to10×175 cm² flasks of sub-confluent BSC-1 cells in 5 ml of serum-freeGibco Earle's Minimal Essential Medium (MEM). The virus was allowed toadsorb for 45 minutes at 37° C. 30 ml of MEM containing 2% (v/v) Gibcofoetal calf serum (FCS) was then added to each flask before returning to37° C. When the infected cells showed full cytopathic effect, the mediumwas discarded. The cells were detached from the flasks, by tapping, into25 ml of PBS. This was centrifuged at 3,000 rpm for 10 minutes at 8° C.The virus containing cell pellet was re-suspended in 6 ml of PBS andfrozen at −80° C. Once thawed, the cell debris were removed bycentrifugation, 3,000 rpm for 10 minutes at 8° C. Aliquots of thesupernatant were frozen at −80° C. Virus was titred by plaque assay.

Plaque Reduction and Neutralisation Assays (PRNT)

The test substances were diluted in serum free MEM and pre-incubated for1 hour at 37° C. with 10⁴ pfu of Lister strain virus particles. Themixture was applied to a monolayer of Vero cells pre-seeded in 24-wellplates. The infected cells were overlayed by addition of 2× MEMincluding 2% carboxymethylcellulose followed by incubation at 37° C.; 5%CO2 for 3 days. The cells were fixated by incubation with PBS; 10%formalin and stained using 20% Ethanol containing 0.1% crystal violetand the number of plaques in each well was recorded by counting.

The EEV-neutralization assay was performed using a similar protocolexcept for that IHD-J strain was used and the carboxymethylcelluloseoverlay was omitted. The IHD-J/Mini-H mixture was pre-incubated with thecells before addition of the Mini-V dilutions and thereby only detectiona Mini-V effect on the EEV mediated virus spreading.

q. In Vivo Protection Assays

The mouse tail lesion model was used to analyze the in-vivo protectionconferred by an anti-VV antibody composition. The experiments wereperformed at the Health Protection Agency, Porton Down, UK (“HPA”). Inbrief, the mice were challenged by injection of infectious vacciniavirus particles of either the Lister or the NYCBOH strain into the tailvein of the mouse. The used amount of virus results in a countablenumber of virus induced lesions on the tail within seven days.Twenty-four hours after or prior to viral challenge, increasing amountsof test antibody compound Mini-V, SymVIG, Sym002 anti-V rp-Ab or anunrelated polyclonal antibody was injected intraperitoneally (I.P.). orintramuscular (LM). In some of the experiments a total of 2.5 mgCidofovir was injected intramuscularly (1.M) as a positive control forviral inhibition.

r. Composition of Sym002 Mix

Antibodies for Sym002 mix was purified individually as described inExample 1, section o, and subsequently mixed. The final antibodyconcentration of Sym002 mix was 1.09 mg/ml and was produced mixing 0.2mg of each of the antibodies 02-029, 02-058, 02-086, 02-113, 02-147,02-186, 02-188, 02-195, 02-197, 02-211, 02-225, 02-229, 02-235, 02-286,02-295, 02-303, 02-339, 02-461, 02-482, 02-488, 02-526, 02-551, 02-586,02-589, 02-607, 02-633, 0.574 mg of 02-037 and 0.171 mg of 02-203. Theantibody identity is given in Table 5.

s. Affinity Measurements of Antibody-Antigen Interactions

Affinities of antibody-antigen interactions were measured by surfaceplasmon resonance using a Biacore 2000 (Biacore AB). The antigen (B5R,VCP, A33R, or A27L) was immobilized on a CM5 chip surface using standardamine coupling chemistry to a level resulting in a RU_(max) ofapproximately 100 RU or less. Purified antibody Fab fragments (seesection 0) were diluted serially in HBS-EP running-buffer (Biacore AB)and passed over the chip at 10-50 μl/min. Rate constants (k_(on) andk_(off)) and affinity constants (K_(D)) were determined using theBIAevaluation software (Biacore AB) by global fitting of four to sixdifferent concentrations passed over the same sensor surface.

t. Preparation of Fab Fragment by Papain Digestion of PurifiedAntibodies

Fab fragments were produced from purified antibodies by using theImmunoPure Fab preparation Kit (Pierce, USA cat. No.: 44885) accordingto manufactures instructions. Briefly, 1.2 mg of the antibodies subjectfor Papain cleavage were dialyzed against 20 mM sodium phosphate, 10 mMEDTA, pH 7 at 4° C. The dialyzed antibody solution were added to 250 μlgel immobilized Papain, pre-equilibrated and suspended in 20 mMCysteine/HCl, 20 mM sodium phosphate pH 7. The reaction were incubatedat 37° C. with shaking over night, followed by centrifugation forremoval of the gel immobilized Papain. Fab fragments were purified bypassing the supernatants through protein A columns. The eluates weredialyzed against PBS and up-concentrated by using Spectrum gel/absorbent(Spectrum Laboratories, Cat. No.: 292600). The Fab fragments wereanalyzed by SDS-PAGE and the concentrations were determined by OD₂₈₀measurements.

Example 2

In the present Example the isolation, screening, selection and bankingof clones containing cognate V_(H) and V_(L) pairs expressed as Fabs orantibodies with anti-vaccinia virus specificity was illustrated.

Donors

Twelve donors were recruited from a smallpox vaccination program ofBritish first line responders in collaboration with the HealthProtection Agency (HPA), United Kingdom. The donors included bothprimary and secondary vaccinia virus-immunized individuals. Blood waswithdrawn in range of 9 to 21 days after vaccination. The B cellfraction was recovered by anti-CD19 MACS column purification and thesub-population of plasma blasts identified by a CD38high andCD45intermediate cell marker expression profile and single-cell sortedby FACS into 96-well plates as described in section a) of Example 1. Thepercentages of plasma blasts expressing anti-vaccinia virus antibodieswere estimated by ELISpot (Example 1, section b). From 0 to 0.6% of thetotal plasma cells were specific, and plasma cells from the top fivedonors (0.3-0.6%) were used to isolate cognate V_(H) and V_(L) codingpairs. All the selected donors belonged to the group of secondaryimmunized donors.

Isolation of Cognate V_(H) and V_(L) Coding Pairs

The nucleic acids encoding the antibody repertoires were isolated fromthe single cell-sorted plasma cells by multiplex overlap-extensionRT-PCR (Example 1, section c). The multiplex overlap-extension RT-PCRcreates a physical link between the heavy chain variable region genefragment (V_(H)) and the full-length light chain (LC). The protocol wasdesigned to amplify antibody genes of all V_(H)-gene families and thekappa light chain, by using two primer sets, one for V_(H) amplificationand one for the LC amplification. Following the reverse transcriptionand multiplex overlap-extension PCR, the linked sequences were subjectedto a second PCR amplification with a nested primer set.

Each donor was processed individually, and 400 to 1200 overlap productswere generated by the multiplex overlap-extension RT-PCR. The generatedcollection of cognate linked V_(H) and V_(L) coding pairs from eachdonor were pooled and inserted into a Fab expression vector as describedin Example 1 section d). The generated repertoires were transformed intoE. coli, and consolidated into ten 384-well master plates and stored.

Screening

Fab-fragments were obtained starting from the master plates, andscreened against inactivated IHD-W and Lister strain as well as againstthe individual recombinant antigen proteins A33R, VCP and B5R asdescribed in Example 1, section e). Several hundred secondary hits weresequenced and aligned. The majority fell in clusters of two or moremembers, but there were also clones that only were isolated once,so-called singletons. Representative clones from each cluster and thesingletons were subjected to validation studies as described in Example1, section e).

A total of 89 clones passed the validation. These are summarized inTable 5. Each clone number specifies a particular V_(H) and V_(L) pair.The IGHV and IGKV gene family is indicated for each clone and specifiesthe frame work regions (FR) of the selected clones. The amino acidsequence of the complementarity determining regions (CDR) of an antibodyor Fab-fragment expressed from each clone are shown, where CDRH1, CDRH2,CDRH3 indicate the CDR regions 1, 2 and 3 of the heavy chain and CDRL1,CDRL2 and CDRL3 indicate the CDR regions 1, 2 and 3 of the light chain(definitions according to Kabat et al. 1991, Sequences of Proteins ofImmunological Interest, 5th edn. US Department of Health and HumanServices, Public Health Service, NIH.). Finally, the antigen specificityof the antibody or Fab-fragment expressed from the V_(H) and V_(L)coding pair contained in the clones is indicated. The antigenspecificity was either identified according to the binding propertiesanalyzed during validation, by binding to in vitro translated antigens,or by Western blotting. The in vitro translated approach identified H3I,A56R, and D8L as target proteins. Un-identified antigens targeted byantibodies produced from the selected clones using Western blotting,were assigned a letter from B to L according to migration properties inSDS-PAGE (Example 1, section g. subparagraph “Characterization of theantigen specificity of the individual antibodies”). Clones indicated asreactive against antigen A in Table 5, were positive in ELISA and FLISAscreens against the IHD-W and/or Lister strain, but did not interactwith a particle associated antigen detectable by Western blotting. Whereno particular antigen is indicated, no further analysis with respect tobinding reactivity has been performed, antibodies expressed from theseclones are however reactive against the IHD-W and/or Lister strain.

The complete variable heavy and light chain sequence can be establishedfrom the information in Table 5. The only exception is clone 02-291,since this clone carry an 11-codon insertion in the so-called hypervariable region 4 (framework region 3) of the heavy chain. For thisreason the complete heavy chain amino acid sequence is given here forthis particular clone:

(SEQ ID NO: 49) QVQLVQSGAEVKKPGYSVKVSCQASGLTFSNYRVSWVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVTISADRSPSRVTSLADKSTITVYMELSSLRSEDTAVYYCARSRGSQDYYGMDVWGQGTTVTVSS.

TABLE 5 CDRH1 CDRH2 CDRH3 IGHV 3   3 5            69       0               0 Clone gene 1abc2345 # 012abc3456789012345 #234567890abcdefghijklmno123 # 02 029 1-24 E---LAMH  50GFDP--EDGEAVYAQGFQG 139 CATDVWRRTPEGGTDWF-------DPW 228 02-031 4-34G---FHWS  51 EIN---HSGSTNYNPSVKS 140 CAGSRSFDLLTAYDLFHRKGNAM-DVW 22902-037 6-1 NNI-ASWN  52 RTYYR-SKWYDDFALSVKG 141CARGDVLRYF--------------DYW 230 02-058 3-30 R---YGMH  53FLSF--DERNKFYPDSLKG 142 CAKGGLGTNEF-------------DHW 231 02-086 5-51T---YWIG  54 IIYP--GDSDTKYSPSFQG 143 CATLPRYDAYGARIR---------DYW 23202-089 1-2 G---HYMH  55 WINP--SSGGTNYAQKFQG 144CARYCSSPTCS-------------IVW 233 02-112 2-5 TSG-VGVD  56LIY---WDDDKRYSPSLKS 145 CAHSSQRVVTGL------------DFW 234 02-113 2-70TSG-MCVS  57 FID---WDDDKYYTTSLKT 146 CARIRTCYPDLYGDYNDAF-----DIW 23502-147 3-7 K---YWMS  58 NINQ--EGSAKHYVDSVKG 147CARAADYGDY--------------VRP 236 02-156 3-30 S---YGMH  59LISY--HGNTKYYADSVKG 148 CAKHVAAGGTL-------------DYW 237 02-159 1-46N---YYIH  60 IINP--SGGSTTYAQRFQG 149 CARVIRKYYTSSNSYLTEQAF---DIW 23802-160* 3-9 D---YAMH  61 GCSW--NSGFISYADSVKG 150CVKETVAGRRGAF-----------DYW 239 02-166 4-34 G---YYWT  62EIN---HSANTDYKPSLKS 151 CARGREWPSNF-------------DSW 240 02-169 5-51T---YWIG  63 IIYP--GDSDTRYSPSFQG 152 CARRGSTYYY--------------DTW 24102-172 3-21 S---YTMN  64 AITS--STTYIYYADSVKG 153CASKPYGGDFG-------------SYW 242 02-183 5-51 K---YWIG  65IIYP--EDGDTRYSPAFQG 154 CARPPSNWDESF------------DIW 243 02-186 1-3N---YAVH  66 WINV--GNGQTKFSQRFQG 155 CARDPTQWLLQGDVYDM-------DVW 24402-188 4-30-4 SGD-YYWN  67 NIY---STGSTYYDPSDQN 156CAREAWLGEPLLLGDDAF------DIW 245 02-189 3-30 K---YYMH  68TISY--DVKNKDYADSVKG 157 CARDGAGEWDLLMRRDF-------DYW 246 02-195 1-18T---YGIS  69 WISA--WDGNTKYGEKFQD 158 CARDPARRPRSGYSVF--------EYW 24702-197 4-4 SN--NWWS  70 EIY---HSGNTNYNPSLQS 159CARDNRQSSSWVEGFFYYYGM---DVW 248 02-201 1-46 K---YYIH  71MINP--SGGTTTYAQKFQG 160 CARLRLGATIGRD-----------DYW 249 02-203 4-30-4RGD-FYWS  72 YIY---YTGSTYYNPSLKS 161 CARDRASSGYDSRVWF--------DPW 25002-205 3-30 D---YTTH  73 IVLY--DGKNKNYADSVRG 162CARTYRVYAKFDPF----------DVW 251 02-211 3-9 D---YAMH  74GISW--NSEYIGYEDSVKG 163 CGKDGVPGRRGYI-----------EDW 252 02-214* 3-9D---YAMH  75 TISW--NSGFIDYADSVKG 164 CVKDNIAGRRGSF-----------DSW 25302-215 1-2 D---YYIH  76 WINP--NFGGTDYAQKFQG 165CARDYIRATGATPSKYFIYYYGM-GVW 254 02-219 1-18 S---YAIA  77WISA--YNGNTDYAQKFQG 166 CARARRVTNSPNNWF---------DPW 255 02-225 4-34G---YYWG  78 EIN---HSGSANYHPSLKS 167 CARAGERSGSGSFVLGRF------DFW 25602-229 3-49 D---YAVS  79 LIRSRHYGAKTQFAASVQG 168CTNTSSLAVA--------------GNW 257 02-232 4-39 SRN-YYWG  80TIY---YTGRTYYNPSLKN 169 CARIPQQRVNYF------------DYW 258 02-235 4-39SSTNYYWG  81 TVY---LSGRAYYNPSLKS 170 CARLPGQRITFF------------DYW 25902-237 6-1 TST-AAWN  82 RTYYR-SRWRNDYAGSVRS 171CARGRRFEDDAF------------DIW 260 02-238* 3-9 D---YAMH  83GINW--NSGNIVYADSVKG 172 CVKDSVAGRRGGF-----------DHW 261 02-242 1-fE---YYIH  84 LVDP--EDGEPIYAEKFQG 173 CATRDGDF----------------DHW 26202-243 5-51 S---YWIS  85 IIYG--GDSDTKYSPSFQG 174CVRHGTRYSFGRSDII--------DIW 263 02-246 5-51 S---YWIA  86IIFP--GDSDTRYSPSFQG 175 CTKTPARGAYGDYIS---------GSW 264 02-250* 3-9D---FAMH  87 GVSW--NSDVINYSDSVKG 176 CAKSTKAVRRGSF-----------DYW 26502-267 1-69 D---YAIS  88 GIIP--VFGTPNYAQQFQG 177CARGGELYEGNGYYSFHYF-----DYW 266 02-269 2-5 STG-VGVG  89LIY---WDDEERYSPSLKN 178 CAHTELAF----------------DYW 267 02-271 1-69S---YAIN  90 SIIP--IFATTNYAQRFQG 179 CARVKGTQNYYGM-----------DVW 26802-274 4-34 G---YYWS  91 EVN---HSGSTNYNPSLRS 180CHYYDSTGYYVS------------DFW 269 02-286 1-18 G---YGIS  92WITY--DKGNTNHAQKFRG 181 CARGVVLIQTILF-----------DYW 270 02-290 1-69N---SAIN  93 GVVP--IYDTSHYAQKFKG 182 CARTVLDSGAYSYY----------DSR 27102-291¤ 1-69 N---YRVS  94 GIIP--IFGTTNYAQKFQG 183CARSRGSQDYYGM-----------DVW 272 02-294 3-23 N---YAMS  95AISG--SGGKTYHAHSVRG 184 CAKLRDSSVYSAYVFRVIF-----DCW 273 02-295 3-11D---NYMN  96 YISS--TSGSIYYADSVKG 185 CATLTVASTY--------------DYW 27402-297 3-9 D---YAMH  97 GLNW--NGANIRYADSVKG 186CVKDTVALLTSRGGCM--------DVW 275 02-302 2-5 TSG-VGVG  98LIY---WDDDKRYSPSLKS 187 CAHSPPHGG---------------DYW 276 02-303 3-30T---YGMH  99 FISS--DGSTKYYADSVKG 188 CAKGLSQALNYYGSGS--------PFL 27702-335 3-30 N---YGIH 100 FISY--DGSKKYYVDSVKG 189CAKDRGVSAWYPRDAF--------DIW 278 02-339 4-59 S---DNWS 101YIY---KTGSTNYNPSLKS 190 CARVPLIEAGITIFAKIGAF----DIW 279 02-349‡ 5-51S---FWIG 102 VTYP--GDSDTRYSPSFQG 191 CARGSPMIKFYF------------DYW 28002-351 3-9 N---YAMY 103 GIIW--NSEYIGYADSVKG 192CARATGAGRRNPL-----------DYW 281 02-431 1-46 S---YYMH 104LINP--SSGTTSYAQNFQG 193 CARPYRSYSSSPQ-----------DYW 282 02-437 4-31SPG-YYWN 105 YIY---YSGSTNYNPSLKS 194 CARYYYSSGPKF------------DYW 28302-446 1-69 S---FAIS 106 SIIP--IFGTAHYAQRFEG 195CARNNRPLGALFGM----------DVW 284 02-461 1-18 T---YGIS 107GIRV--HNGNTNYAQKFQG 196 CARGGFNRLV--------------DPW 285 02-482 4-31SAG-YYWS 108 YIH---YTGTTYYNPSLKS 197 CARNIGIYLGGSPGGIRNNWF---DPW 28602-488 5-51 S---YWIG 109 IIYP--GDSDTRYSPSFQG 198CARQQAKTLYYDSSGSKSAF----DIW 287 02-515 4-31 SGG-YYWS 110YIH---YSGSTYYMPSLKS 199 CARVRGNIVATTAFYYYYGL----DAW 288 02-516 3-11D---YYMS 111 YTNL--FTGYTNYADSVKG 200 CAKFDYGEGAYHF-----------DFW 28902-517 4-31 GA--YHWS 112 YIY---YTGNTYFNPSLKS 201CARDPIALPGRGVF----------DYW 290 02-520 4-34 A---YYWS 113EIS---HSGSTHYNPSLNS 202 CSSGYYFAGGEF------------DYW 291 02-526 3-49D---YTMS 114 FIRGKKFGGTKDYAASVKG 203 CTRDRGYSDHTGLYTRFGF-----DSW 29202-532 1-69 D---HSIG 115 KIIP--IYGRANYAQKFQG 204CARWRGGYSGYGDYF---------DSW 293 02-536 4-4 SS--HWWN 116EIY---HSGSTNYNPSLKS 205 CARDPQKPRQHLWPNPYYYSGM--DVW 294 02-551 1-69Y---YAIN 117 GIVP--MVGPADYAEKFRG 206 CARGRSWRGYL-------------DYW 29502-559 1-46 N---YYMH 118 LINP--SGDSTTNAQKFQG 207CARDYGDYCGGDCPYDAF------DIW 296 02-572 1-69 S---FGIS 119GIIP--IFGTPNYSLKFQD 208 CARDKGESDINGWQTGAFYYGM--DVW 297 02-575 2-26NDR-MGVS 120 HIF---SNDERSHSSSLKS 209 CARIDSVGWPSSHYYGM-------DVW 29802-586 4-59 S---YYWS 121 YIF---YSGNTNYNPSLKS 210CARDRITGYDSSGHAF--------DIW 299 02-589 4-34 G---YYWT 122EIN---QNGRSNHNPSLKS 211 CARGGKFCGSTSCFTEGRL-----DYW 300 02-607 3-30S---YGMH 123 VISY--DGRYKFYANSVKG 212 CAKDSGRYSSLGHYYYYGM-----DVW 30102-611 5-51 N---YWIG 124 IIHP--GDSETRYSPSFQG 213CARGYYYDTSGYRPGSF-------QHW 302 02-612 3-30 T---YTMH 125VISY--DGTNKYHTDSVKG 214 CARPLFYGAGDAF-----------DIW 303 02-614 1-69N---YAII 126 EIIP--KFGTANYAQKFQG 215 CADWVVGNYNGL------------DVW 30402-617 1-46 N---YYVH 127 LINP--SAGKTTYAQRFQG 216CAREGKHDFWRGYFSPLGM-----DVW 305 02-621 1-69 S---HGVN 128GIIP--VFGTTNYAQSLQG 217 CATARNSSNWYEGHYYL-------AHW 306 02-626 4-30-4TGD-YYWS 129 YVF---NSGSTYYNPSLQS 218 CANMVVVATQPKNWF---------DPW 30702-628 4-30-4 SGY-YYWN 130 YID---YRGTTYYSPSFKS 219CASYGSGMGSEYYF----------GHW 308 02-632 1-2 G---YYIH 131RINP--ITDVTNYAQIFQG 220 CGRVGREAFYYYGM----------DVW 309 02-633 1-2A---YYIH 132 RINP--DSGGTDFSQKFQG 221 CARASRRLTTHNYF----------DGW 31002-634 3-48 T---YEMS 133 YIGS--GGVTIYYADSVKG 222CARVRGGRYF--------------DYW 311 02-640 5-51 T---YWIA 134IIWP--VDSDTRYSPSFQG 223 CASGSGYDSYYNM-----------DVW 312 02-643 7-4-1S---YAMN 135 WINT--NTGNPTYAQGFTG 224 CARDSSTVTGLMTEYNWF------DPW 31302-649 4-31 SGP-YYWS 136 YSS---NRGIAYYNPSLKS 225CATEKGSGGDVGKF----------DNW 314 02-650 1-69 S---NPVS 137GIIP--FAQKVLGAQRVRD 226 CATGQQLYSL--------------HYW 315 02-651 1-2D---YYLH 138 RINP--KSGDTHHVQKFQG 227 CAREGPQFYYDSGDYYSAHSPGDFDHW 316CDRL1 CDRL2 CDRL3 IGKV 2     3          3 5 8 9 Clone gene45678901abcdefghi234 # 0123456 # 89012345a678 # 02-029 3-11RASQSVRR---------SLA 317 DASNRAT 406 CLQRSNWP-ITF 495 02-031 1-39RASQGISN---------SLN 318 GASGLES 407 CQQSYRTL-YTF 496 02-037 1-5RASQSISI---------WLA 319 KASTLES 408 CQQYNGYSEVTF 497 02-058 1-5RASQSIGN---------WLA 320 DASSLKS 409 CQQYDTYP-ITF 498 02-086 1D-33QASQDISK---------YLN 321 DASNLET 410 CQQYDNLP-PTF 499 02-089 3-20RASESVRSN--------YLA 322 GASSRAT 411 CQQYGRSP-LTF 500 02-112 1-39RASQSIST---------YLN 323 AASSLQS 412 CQQSYNTP-ATF 501 02-113 1D-33QASQDIKY---------YLN 324 DASNLET 413 CQQYENVP-YTF 502 02-147 2-28RSSKSLLHSNGYN----YLD 325 LASNRAS 414 CMQALQIP-RTF 503 02-156 2-24RSSESLVNNDGNT----YLS 326 KISNRFS 415 CMQTTHIP-HTF 504 02-159 1-39RASQNISN---------FLL 327 AASSLQS 416 CQQTYGNP-LTF 505 02-160* 1-39RASQSINN---------YLN 328 AVSSLQT 417 CQQSFRTP-HTF 506 02-166 3-11RASQSVDR---------YLN 329 DASNRDT 418 CQQRAIWP-PEF 507 02-169 1-39RASQSIWT---------FLN 330 TASSLQS 419 CQQSFTSW-WTF 508 02-172 1-5RASQSISN---------WLA 331 KASNLES 420 CQQYSNYP-ITF 509 02-183 1-17RASQDISN---------DLG 332 LASSLQS 421 CLQHNSF--LTF 510 02-186 2-30RSSQSLVYSDGNT----YLH 333 KVSNRDS 422 CMQGTHWP-PAF 511 02-188 1-17RASQGIGY---------DLG 334 AASSLQS 423 CLQLHTFP-RTF 512 02-189 1-39RASQSISN---------YLS 335 AASLLQT 424 CQQGYSTP-YTF 513 02-195 3-15RASQSVSS---------NLA 336 GASTRAT 425 CHQYNYWPPLAF 514 02-197 3-20RASQSIASA--------YLA 337 GASSRPT 426 CQQYGISP-RTF 515 02-201 2-28RSSQSLLHSNGYN----YLD 338 LGSTRAS 427 CMQALQTP-HTF 516 02-203 ID-33QASHDVSN---------FLN 339 DASNLKT 428 CHQYDSLP-FTF 517 02-205 3-20RASQSVSSN--------YIA 340 GASSRAT 429 CQQFGYSPRFTF 518 02-211 1-39RASQSIRT---------YLN 341 AASSLQS 430 CQQTYITP-KSF 519 02-214* 1-39RASQTIST---------YLN 342 AASSLQS 431 CQQSYRTP-LTF 520 02-215 1-27RASQGISN---------YLA 343 GASTLQS 432 CQKYDSAP-YTF 521 02-219 1-39RASRSIST---------YLN 344 AASSLQS 433 CQQTYTIP-LTF 522 02-225 1-39RASQSIHT---------YLN 345 TASNLQS 434 CQQSYSTL-RTF 523 02-229 4-1KSSQSVLYSSNNNN---YLA 346 WASTRES 435 CQQYYKTP-PTF 524 02-232 1-5RASQTIST---------WLA 347 DASSLES 436 CQQYNSYP-LTF 525 02-235 1-5RASQSIST---------WLA 348 DASSLES 437 CQQYNFY--GTF 526 02-237 1-39RASQSISN---------YLN 349 GASSLES 438 CQQSYSIP-RTF 527 02-238* 1-39RASLNIRN---------YLN 350 AASTLQI 439 CQQSYSMSPYTF 528 02-242 1-16RASQVIGK---------YLA 351 ATSILQS 440 CQQYNSFP-LTF 529 02-243 1-17RASQGIRN---------DLG 352 AASSLQS 441 CLQQNNYP-WTF 530 02-246 1D-33QASHDINK---------YLN 353 DASNLET 442 CQQYDNFP-YTF 531 02-250* 1-39RASQSINN---------YLN 354 AASSLHS 443 CQQTYIST-RTF 532 02-267 1D-33QASQDISN---------YLN 355 DASHLET 444 CQQYDNLP--LF 533 02-269 1D-33QASQDISF---------YLN 356 DASILET 445 CQQYDNLI--TF 534 02-271 1-5RASQSISS---------WLA 357 KVSSLES 446 CQQYESDI-FTF 535 02-274 1-16RASQDISN---------YLA 358 AASSLLS 447 CQQYGRYP-LTF 536 02-286 1-39RASQSVST---------FLN 359 GVSNLQS 448 CQQSHRTP-YTF 537 02-290 1-5RASQDVSP---------WLA 360 KASSLES 449 CQQYQTY--STF 538 02-291¤ 1-5RASQGISD---------WLA 361 KASSLES 450 CQQYESDS-WTF 539 02-294 1D-33QASQDISN---------YLN 362 DTSNLET 451 CQQYDNLP-FTF 540 02-295 2-30RSSQSVVYSDGNI----YLN 363 QVSNRDS 452 CMQGTHWP-YSF 541 02-297 1-12RASQDISS---------WLA 364 AASSLQS 453 CQQAYSFP-WTF 542 02-302 1D-33QASQDISN---------YLN 365 DASNLET 454 CQQYDNL--PTF 543 02-303 3-20RASQSVSSL--------YVG 366 GTSSRAT 455 CQQYGTSP-WTF 544 02-335 1-39RASQSISS---------FLN 367 GATTLQS 456 CHQSYSLP-FTF 545 02-339 3-20RASQSVSS--------SYLA 368 RASSRAA 457 CQQYVASP-FTF 546 02-349‡ 3-20RASQSVSSRASQSVSSNYLA 369 GASTRAA 458 CHQYGTSP-RTF 547 02-351 1-39RASQTIRN---------YLN 370 TASSLHS 459 CQQSYITP-YTF 548 02-431 3-20RASQSVSNN--------NLA 371 GASSRAA 460 CQQYGSSP-YTF 549 02-437 1-12RASQGISN---------WLA 372 AASSLQS 461 CQQANSFP-FTF 550 02-446 1-9RASQGIGG---------ALA 373 AASTLQS 462 CQQLDTYP-LTF 551 02-461 3-20RASQSVSSN--------YLA 374 GASSRAT 463 CQQYASSP-YTF 552 02-482 1-39RASQSISR---------HLN 375 AASSLQT 464 CQHSSKTP-FTF 553 02-488 1-5RASQSIST---------YLA 376 KASSLEP 465 CQQYSSY--LSF 554 02-515 1-12RASQGVSN---------WVA 377 AASSLQS 466 CQQANGFL-WTF 555 02-516 1D-12RASQGIST---------FLA 378 AASSLQS 467 CQQAHSFP-VTF 556 02-517 3-20RASQSVTSN--------YLA 379 GASNRAT 468 CQQYGSSP-LTF 557 02-520 1-12RASQGIST---------WLA 380 AASTLQH 469 CQQANSFP-RTF 558 02-526 2-28RSSQSLLHSNGYN----YLD 381 LGSNRAS 470 CMQSLQT--VTF 559 02-532 1-39RASQSISN---------YLN 382 AASRLQS 471 CQHSYETPPYTF 560 02-536 1-5RASQSLNN---------WLA 383 DASSLQS 472 CQQYNFYP-WTF 561 02-551 3-20RASQSVSNN--------YLA 384 GASSRAT 473 CQQYGGSP-QTF 562 02-559 1-27RASQGIFN---------YLA 385 GASTLRS 474 CQKYNSAP-LTF 563 02-572 1-12RASQNIGN---------WLA 386 SASSLQN 475 CQQANSFP-VTF 564 02-575 1-12RASQDIIS---------WLA 387 AASSLQS 476 CQQTHSFPPWTF 565 02-586 1-5RASQSIYI---------WLA 388 DASSLES 477 CQQYHHYS-PTF 566 02-589 1-39RASQSISR---------SLN 389 AASTLQS 478 CQQSYSTL-RTF 567 02-607 1-39RASQPISS---------FLN 390 AASSLQS 479 CQQGYSTP-PTF 568 02-611 1-5RASQSISS---------WLA 391 HAFSLEG 480 CQQYDSYP-YTF 569 02-612 1-39RASQSFNG---------YLN 392 AASTLQS 481 CQQSYSTP-RTF 570 02-614 3-20RASQTVIST--------YLA 393 GASSRAT 482 CQQYSDS--LTF 571 02-617 3-20RASQSVSSG--------SLD 394 GASNRAS 483 CHQYGGAQ-GTF 572 02-621 1D-33QASQDISN---------YLN 395 DASNLET 484 CQQYDTLPPITF 573 02-626 1-39RASQSISS---------YLN 396 AASSLQS 485 CQQSHSSP-WTF 574 02-628 1-39RASQSIRS---------YLN 397 GASSLQS 486 CQQSYLAP-WTF 575 02-632 1-9RASQGISS---------YLV 398 AASTLES 487 CQQFNNYP-YTF 576 02-633 1-16RASQAISN---------YLV 399 GAFILES 488 CQQYHTYP-FTF 577 02-634 3-20RASQSVSST--------YLA 400 GASNRAT 489 CQKYGRSPTWTF 578 02-640 1-39RASQSISN---------HLN 401 VASSLQG 490 CQQGFTTP-ITF 579 02-643 1-39RASQSISS---------YLN 402 AASSLQS 491 CQQSYSTP-YTF 580 02-649 1-39RANQSIDD---------YLH 403 DASTLHS 492 CQQSYTIPLWTF 581 02-650 2-30RSSQSLVYADGDT----HLN 404 HVSNRDA 493 CMQGTHWP-PTF 582 02-651 1-39RASQSITN---------CLN 405 GASTLQS 494 CQQSDSTP-YTF 583 IGHV and IGKV genenames were assigned according to the official HUGO/MGT nomenclature(IMGT; Lefranc & Lefranc, 2001, The Immunoglobulin FactsBook, AcademicPress). Numbering and alignments are according to Chothia (Al-Lazikaniet al. 1997 J. Mol. Biol. 273: 927-48). *These clones were isolated fromdifferent donors but utilize highly similar VDJ and VJ rearrangements.¤These two clones carry an 11-codon insertion in the so-calledhypervariable region 4 (framework region 3). ‡This clone carries aneight-codon insertion in CDRL1.

Mirroring the Humeral Immune Response

In order to illustrate that the cognate V_(H) and V_(L) coding pairsisolated and selected as described above mirrors the natural humeralimmune response raised upon challenge with vaccina virus, serum samplesfrom ten of the twelve donors were tested for reactivity against threevirus strains (Lister, IHD-W and IHD-J), and five recombinant antigens(B5R, VCP, A27L, A33R and L1R). The antibody titers were determined asthe minimum sera dilution required for a four-fold background signal andthe determined values are displayed in FIG. 7. The titers varied amongthe different antigens and donors, this is likely to reflect differencesin immunogenicity, efficient up-take of the vaccinia virus, time fromvaccination to blood withdrawal, primary and secondary response, etc.Comparison of the specific antibody titers from the donors andidentified cognate Fabs/antibodies with the same reactivity, suggestedthat the antibody diversity recovered in the present experiment includedthe specificities present in the collected serum samples. The analysisthereby supports the claim that the identified anti-vaccinia virusrepertoire reflects the specificity of the natural humeral immuneresponse.

The vaccinia virus-specific clones also display a high diversity. Allimmunoglobulin variable region heavy chain gene families (IGHV),including IGHV7, as well the immunoglobulin kappa light chain genefamilies (IGKV) 1-4, were found among the validated clones. The V-genefamilies for each isolated and selected clone are indicated in Table 5.The heavy chain V-gene usage correlated with what has been foundpreviously in human antibody repertoires, with a frequent usage of theIGHV3, IGHV1 and IGHV4 families. The light chain V-gene usage wasdominated by the IGKV1 family, mainly due to the frequent usage of theIGKV1-39 gene. This gene is also among the most frequently used lightchain V-genes in the human immunoglobulin repertoire. As a whole, theIGKV1 genes make up 75% of the repertoire. The only other IGKV gene ofprominent usage in the isolated repertoire was the IGKV3-20, which alsois the most frequently expressed light chain gene in humans. The IGKVfamilies 5 and 6 were the only families not represented in therepertoire, IGKV5 and 6 are very rarely utilized in a human antibodyresponse, or may not even consist of functional IGKV genes (de Wildt etal. 1999, J. Mol. Biol. 285:895-891; Lefranc & Lefranc 2001, TheImmunoglobulin FactsBook, Academic Press). In conclusion, the diversityof the isolated repertoire is evidence of an unbiased recovery ofV-genes by multiplex overlap-extension RT-PCR, indicating that theisolated antibodies minor the diversity of the humeral response inhumans.

Further, due to the cognate pairing of the V_(H) and V_(L) codingsequences representing the V_(H) and V_(L) pairing originally present inthe donor from which such a cell is derived, the affinity of the humeralimmune response raised upon vaccination with vaccinia virus is alsoconsidered to be mirrored by the isolated antibodies.

Generation of a Cell Bank of Individual Antibody Expressing Members

A subset of 47 unique cognate VH and VL coding pairs corresponding toclone nr 02-029, 02-031, 02-037, 02-058, 02-086, 02-089, 02-112, 02-113,02-147, 02-156, 02-159, 02-160, 02-169, 02-172, 02-186, 02-188, 02-195,02-197, 02-201, 02-203, 02-205, 02-211, 02-225, 02-229, 02-232, 02-235,02-243, 02-271, 02-286, 02-295, 02-297, 02-303, 02-339, 02-431, 02-461,02-482, 02-488, 02-516, 02-520, 02-526, 02-551, 02-586, 02-589, 02-607,02-628, 02-633 and 02-640 of Table 5 were selected for expression ascomplete antibodies. The VH and VL coding pairs were selected from the89 validated antibodies, according to the Fab reactivity in ELISA,Western blotting, antigen diversity, and sequence diversity. Theselected cognate V-gene pairs were transferred to a mammalian expressionvector. The transfer process is described in Example 1 section f). Theindividual expression constructs were co-transfected with aFlp-recombinase expressing plasmid into the CHO-FlpIn recipient cellline (Invitrogen), followed by antibiotic selection of integrants. Thetransfections, selection, and adaptation to serum free culture wasperformed as described in Example 1, section g). The adaptation processwas continued until the doubling time was below 32 hours. This was onaverage completed within a 4-6 week period; thereafter the individualcells lines were banked.

Example 3

In the present Example the biological activity of a mixture ofmonoclonal anti-VV antibodies was compared to a serum derived VIGproduct.

The first sixteen individual cell lines introduced into the bankgenerated in Example 2, were used to express monoclonal antibodies,which where purified, and characterized individually and mixed togenerate the Mini-V preliminary antibody composition, a compositionwhich was composed of a mixture of 16 monoclonal antibodies containingthe cognate V_(H) and V_(L) pairs corresponding to clone nr 02-029,02-031, 02-037, 02-058, 02-086, 02-089, 02-112, 02-113, 02-147, 02-156,02-159, 02-160, 02-169, 02-172, 02-211 and 02-243 of Table 5 in Example2. The Mini-V composition was produced to resemble a polyclonal antibodyproduct, to verify the biological activity of such a product. Inparallel, an oligoclonal composition was compiled consisting of threeantibodies specific for IMV antigens as indicated by Western blotting.This oligoclonal antibody composition was termed Mini-H, and wascomposed of a mixture of 3 monoclonal antibodies corresponding to clonenr 02-31, 02-211 and 02-243 of Table 5.

The specific binding to inactivated Lister strain of the Mini-V andMini-H compositions were compared to the serum derived antibodies of thefive processed donors, which had been affinity purified using Protein-Acolumns (termed SyrnVIG) (FIG. 8). The direct binding analysesdemonstrated at least a 100-fold increase in the specific bindingactivity of the recombinant antibody compositions.

The neutralizing activity of the two compositions was assayed in vitroby plaque reduction and neutralization assay as described in Example 1,section p). The anti-viral potency was indicated as the antibodyconcentration required for 50% reduction of viral infectivity detectedas number of plaques caused by a vaccinia virus infection of a confluentmonolayer of adhered cells (IC₅₀). Mini-V showed a 100-fold increasedpotency compared to SymVIG (Table 6). This correlated with the profoundspecific activity of Mini-V. Interestingly, Mini-H was 10-fold lesspotent than Mini-V, indicating superiority of a polyclonal antibodycomposition, including reactivity against both IMV- and EEV-particles.

TABLE 6 IMV- EEV- Antibody neutralization neutralization composition(μg/ml) (μg/ml) Mini-V 0.5  25 Mini-H 6.25 n.a. SymVIG 62.5 250

EEV-specific infection was detected in a plaque reduction assay asdescribed in Example 1, section p) in the presence of 100 μg/ml of theMini-H IMV-neutralizing antibody composition. The EEV-neutralizationpotency of Mini-V was only found to be 10-fold more profound than SymVIG(Table 6). The Mini-V composition only contains two EEV-specificantibodies. This is most likely the reason that the mini-V compositionhas a lower anti-EEV than anti-IMV activity, and this suggests that anincreased proportion of anti-EEV antibodies will improve theEEV-neutralization.

The effect on vaccinia virus replication of Mini-V in vivo has beeninvestigated using the mouse tail lesion model (see Example 1, sectionq). Both SymVIG and Mini-V significantly reduced the number of taillesions (FIG. 9). A nearly complete elimination of pocks was observedwith the 100 μg to 300 μg Mini-V doses, in contrast to a maximum of ˜65%inhibition observed for the highest concentration of SymVIG. Theseexperiments suggest that the in vivo specific activity of Mini-V is atleast 100-fold higher than SymVIG, correlating with the in vitroneutralization and binding studies.

Example 4

The present Example illustrates the generation of a recombinantpolyclonal anti-VV antibody and compares the biological activity of ananti-VV rpAb with that of a mixture of monoclonal antibodies and a serumderived polyclonal VIG product.

Generation of a Polyclonal Cell Line and Production of Anti-VV rpAb

The aim of the lead identification for the production of the recombinantpolyclonal anti-VV antibody was to select a composition with thebroadest possible reactivity against vaccinia virus and thus presumablyalso variola virus. The individual antibody members included in theanti-VV recombinant polyclonal antibody were selected based on severalcharacteristics:

-   -   Antigen reactivity; in principle all antigen reactivities        identified in Example 2 should be represented in the        composition, and the reactivity should be specific and of high        affinity. Further, if possible an average of 2-3 antibodies        against each identified antigen should be included.    -   Biochemical properties; only antibodies with well-defined        characteristics were included in the final product. The        individually expressed antibodies were analyzed by reducing and        non-reducing SDS-PAGE, and ion exchange-profiles. Antibodies        with an aberrant biochemical behavior were subjected to detailed        analysis and most likely excluded from the final composition.    -   Cell line characteristics; to optimize the stability of the        polyclonal cell line, individual cell lines with similar        doubling times, as well as productivity after adaptation were        preferred.

Twenty eight individual cell cultures expressing cognate V_(H) and V_(L)coding pairs corresponding to clone nr. 02-029, 02-037, 02-058, 02-086,02-113, 02-147, 02-186, 02-188, 02-195, 02-197, 02-203, 02-211, 02-225,02-229, 02-235, 02-286, 02-295, 02-303, 02-339, 02-461, 02-482, 02-488,02-526, 02-551, 02-586, 02-589, 02-607 and 02-633 of Table 5 in Example2, were selected for the generation of a polyclonal cell line. Theindividual cell lines were thawed and expanded at 37° C. in serum freemedium in shaker flasks to reach at least 4×10⁸ cells of each clonehaving a population doubling time of 21-34 hours. The viabilities werein the range of 93% to 96%, prior to mixing of the individual celllines. Further details to the generation of the polyclonal cell line andthe establishment of a polyclonal working cell bank (pWCB) are found inExample 1, section g).

The recombinant polyclonal anti-VV antibody was produced in laboratorialscale bioreactors as described in Example 1, section n) and purified asdescribed in section o). Three batches have been produced independentlyfrom three vials from the pWCB.

The diversity of the anti-VV rpAb produced from the polyclonal cell linewas analyzed by IEX chromatography using a PolyCAT column; buffer A: 25mM sodium acetate, 150 mM sodium chloride, pH 5.0; B: 25 mM sodiumacetate, 0.5 mM sodium chloride, pH 5.0 (gradient: 30-100% B). The IEXprofiles of the three anti-VV rpAb batches were highly similar.Assignment of the individual peaks in the IEX profile of the anti-VVrpAb product was conducted by running the individual antibodies producedin Example 2, under identical conditions to identify the position of thepeaks in the IEX profile (FIG. 10). Antibodies from two of theindividual cell lines (clones 02-113 and 02-225) included in thepolyclonal cell line were not found in the anti-VV rpAb. These clonesproduce antibodies which are reactive against VCP and antigen L,respectively. However, since there are other antibodies with similarreactivity in the composition the loss of these clones are consideredacceptable for the further analysis.

Biological Activity of the Anti-VV rpAb

The reactivity of the three anti-VV rpAb batches against differentvaccinia virus strains and the recombinant antigens was compared byELISA (FIG. 11). In correlation with the highly similar IEX profiles,the three batches exert a nearly identical binding reactivity againstall the tested antigens supporting a consistent antibody mixture of theproduced polyclonal antibody. For this reason it was decided to pool thethree batches. Further, the anti-VV rpAb batches were compared to SymVIGand the commercial available serum derived VIG product (Cangene). Incomparison to the serum derived VIG, a ˜250 fold increase specificbinding activity of the anti-VV rpAb batches was observed more or lessindependent of the target antigen.

The SYM002 anti-VV rpAb was tested in two types of plaque reduction andneutralization assays (PRNT; Example 1, section p). SYM002 anti-VV rpAbexerted a 40-fold improved specific antiviral activity as compared tothe Mini-V of Example 3 and 800-fold improved specific activity than thecommercial available blood derived VIG product (Cangene) (Table 6). Thesuperior antiviral activity of Sym002 anti-VV rpAb was also observed inthe modified PRNT assay detecting EEV-mediated infection. The IC50values obtained in the in vitro assays are connected with a high degreeof uncertainty, but the presented data clearly demonstrate a superiorspecific activity of Sym002 anti-VV rpAb.

TABLE 6 IMV- EEV- Antibody neutralization neutralization composition(μg/ml) (μg/ml) Sym002 anti-VV 0.125 2 rpAb Mini-V 5.0 10 VIG (cangene)100 200

The Sym002 anti-VV rpAb has been tested for in vivo antiviral activityusing the tail lesion model challenged with either Lister of NYCBOHvaccinia virus strain. The Sym002 pre-lead product was administeredintramuscularly (I.M) 24 hours prior (prophylactic) or post(therapeutic) virus challenges. All data sets demonstrated anapproximately 300-fold increase specific activity of Sym002 anti-VV rpAbas compared to blood derived commercial available VIG (FIG. 12). It wasanticipated that prophylactic administered Sym002 anti-VV rpAb wouldelicit a profound anti-virus effect and not as observed a nearlyidentical antiviral activity. The result is likely to reflect thelimitation of the used mouse model, in which a huge amount of infectiousvirus particles (˜10⁵ PFU) is injected into the tail vein and therebycreating a temporary high local concentration that are giving raise toapproximately 50-70 countable pocks in the untreated situation. In thisscenario the temporary high concentration of virus is likely to exceedthe concentration of administered antibody which thereby is more likelyto influence continues spreading of virus than blocking the primaryinfection. If this is the dynamic of the mouse tail lesion model noprofound potency of Sym002 anti-VV rpAb can be obtained by aprophylactic administration of antibody correlating with theobservation. Combined the in vitro and in vivo data provide evidence forthat all essential antibody reactivities required for in vitroneutralization of vaccinia virus are included in anti-VV rpAv.

A Sym002 mix was compiled by mixing of individual purified antibodies(Example 1, section i) to obtain a more equal representation of eachantibody than present in the anti-VV-rpAb produce from the pWCB (Example4). The anti-viral activity of the two polyclonal antibody compounds wasassayed in the mouse tail lesion model as described above except forthat the indicated doses of antibodies were administered intraperitoneal(LP) 24 hours post challenge with the NYCBOH vaccinia virus. Within thetested dose range we observed an identical antiviral activity of theSYM002 anti-VV-rpAb and the Sym002 mix (FIG. 13) proving that the usedproduction method generate fully biological active polyclonal antibodyproduct.

Example 5 Affinity of Selected Antibodies

The binding kinetic of antibodies can be detected by surface plasmonresonance measurements e.i. by BIAcore. This method requires chemicalimmobilization of the antigens on a CHIP surface, restricting theanalysis to the Sym002 antibodies interacting with A27L, A33R, VCP andB5R, since only these antigens were available as recombinant proteins.Fab fragments were generated from purified IgG1 antibodies by papaindigestions (Example 1, section J). The kinetic measurements revealedaffinities constants (K_(D)) between 10⁻⁸ to 10⁻¹⁰ M⁻¹ of the majorityof the investigated antibodies (FIG. 14). The observed affinities areapproaching the theoretical antibody affinity ceiling (KD at 10⁻¹⁰ M-1)(Foote and Eisen, 1995, Proc Natl Acad Sci USA, 92: 1254-1256)supporting that the selected antibodies repertoire mirrors the naturalhumeral immune response. In addition to the reported data, some of thetested antibodies did not interact with the immobilized antigen. Theexplanation to this observation is unknown but it might be due tosterical constrains introduced by the immobilization or that thestructure of some epitopes may be denatured during either during theimmobilization or the chip cleaning procedures,

1. An anti-orthopoxvirus recombinant polyclonal antibody comprisingdistinct members which in union are capable of binding at least threeorthopoxvirus related antigens.
 2. The anti-orthopoxvirus recombinantpolyclonal antibody according to claim 1, wherein at least two distinctepitopes on the same orthopoxvirus related antigen are bound by saidpolyclonal antibody.
 3. The anti-orthopoxvirus recombinant polyclonalantibody according to claim 1, wherein the distinct antibody membersmirror the humeral immune response with respect to diversity, affinityand specificity against antigens associated with one or moreorthopoxviruses.
 4. The anti-orthopoxvirus recombinant polyclonalantibody according to claim 1, wherein the distinct antibodies areencoded by nucleic acid sequences obtained from one or more donors whohave raised a humeral immune response against an orthopoxvirus.
 5. Theanti-orthopoxvirus recombinant polyclonal antibody according to claim 4,wherein the donors have been vaccinated with a vaccinia virus strain orare recovering from an orthopoxvirus infection.
 6. Theanti-orthopoxvirus recombinant polyclonal antibody according to claim 4,wherein the one or more donors are human, and the polyclonal antibody isa fully human antibody.
 7. The anti-orthopoxvirus recombinant polyclonalantibody according to claim 3, wherein the distinct antibody memberscomprise V_(H) and V_(L) pairs originally present in the one or moredonors.
 8. The anti-orthopoxvirus recombinant polyclonal antibodyaccording to claim 3, wherein the specificity of the individual membersof the anti-orthopoxvirus recombinant polyclonal antibody are selectedsuch that the antibody composition collectively binds antigens thatelicit significant antibody responses in mammals.
 9. Theanti-orthopoxvirus recombinant polyclonal antibody according to claim 1,which comprises binding reactivity against Intracellular Mature Virions(IMV) as well as Extracellular Enveloped Virions (EEV) specificantigens, where the binding reactivity is characterized by distinctmembers capable of binding either an IMV or an EEV specific antigen. 10.The anti-orthopoxvirus recombinant polyclonal antibody according toclaim 9, which comprises binding reactivity against antigens selectedamong the IMV viral proteins A27L, A17L, D8L and H3L and antigensselected among the EEV viral proteins A33R and B5R.
 11. Theanti-orthopoxvirus recombinant polyclonal antibody according to claim10, where the group of antigens from IMV additionally comprises theviral protein L1R and the group of antigens from EEV additionallycomprises the viral protein A56R.
 12. The anti-orthopoxvirus recombinantpolyclonal antibody according to claim 1, where said polyclonal antibodycomprises at least one individual antibody member with bindingreactivity against an orthopoxvirus related regulators of complementactivation (RCA) protein encoded by an orthopoxvirus.
 13. Theanti-orthopoxvirus recombinant polyclonal antibody according to claim12, wherein the RCA binding reactivity is directed to a protein selectedfrom the group consisting of VCP, SPICE, IMP, MPXV-VCP and CMLV-VCP. 14.The anti-orthopoxvirus recombinant polyclonal antibody according toclaim 12, which comprises binding reactivity to at least two of the RCAdomains selected from the group consisting of SCR2, SCR4 and thejunction between the SCR3 and 4 domains.
 15. The anti-orthopoxvirusrecombinant polyclonal antibody according to claim 3, which additionallycontains one or more distinct antibodies encoded from V_(H) and V_(L)pairs selected from one or more donors which have been immunized orvaccinated with a particular orthopoxvirus related antigen. 16-18.(canceled)
 19. A method for treatment or prevention of adverse sideeffects of vaccination with vaccinia virus in a human or an animal,wherein an effective amount of the anti-orthopoxvirus recombinantpolyclonal antibody according to claim 1 is administered to said humanor animal.
 20. A method for treatment or prophylaxis of an orthopoxvirusinfection in a human or animal, wherein an effective amount of theanti-orthopoxvirus recombinant polyclonal according to claim 1 isadministered to said human or animal.
 21. (canceled)
 22. Apharmaceutical composition comprising as an active ingredient, ananti-orthopoxvirus recombinant polyclonal antibody according to claim 1and a pharmaceutically acceptable excipient.
 23. A screening procedurefor selecting V₁₄ and V_(L) sequence pairs capable of encoding a broaddiversity of anti-orthopoxvirus antibodies comprising: (a) expressing anantibody or antibody fragment from a host cell transfected with ascreening vector comprising a distinct member of the repertoire of V_(H)and V_(L) coding pairs, (b) contacting said antibody or antibodyfragment with at least two different vaccinia virus strains and one ormore antigens selected from the group consisting of A27L, A17L, D8L,H3L, L1R, A33R, B5R and VCP in parallel, (c) repeating step a) and b)for each V_(H) and V_(L) sequence pair in the repertoire of sequencepairs, and (d) selecting the V_(H) and V_(L) sequence pairs encoding anantibody or antibody fragment which bind to at least one of the vacciniavirus strains or one of the antigens.
 24. The screening method accordingto claim 23, wherein the V_(H) and V_(L) coding pairs are cognate pairs.25. The screening method according to claim 23, wherein the screeningprocedure does not utilize phage display.
 26. A method for generating arepertoire of V_(H) and V_(L) coding pairs, where the members mirror thegene pairs responsible for the humeral immune response upon challengewith an orthopoxvirus, comprising: (a) providing a lymphocyte-containingcell fraction from a donor vaccinated with an orthopoxvirus orrecovering from an orthopoxvirus infection; (b) optionally enriching Bcells or plasma cells from said cell fraction; (c) obtaining apopulation of isolated single cells, comprising distributing cells fromsaid cell fraction individually into a plurality of vessels; (d)amplifying and effecting linkage of the V_(H) and V_(L) coding pairs, ina multiplex overlap extension RT-PCR procedure, using a template derivedfrom said isolated single cells; and (e) optionally performing a nestedPCR of the linked V_(H) and V_(L) coding pairs.
 27. The method accordingto claim 26, wherein the linked V_(H) and V_(L) coding pairs aresubjected to a screening procedure comprising: (a) expressing anantibody or antibody fragment from a host cell transfected with ascreening vector comprising a distinct member of the repertoire of V_(H)and V_(L) coding pairs, (b) contacting said antibody or antibodyfragment with at least two different vaccinia virus strains and one ormore antigens selected from the group consisting of A27L, A17L, D8L,H3L, L1R, A33R, B5R and VCP in parallel, (c) repeating step a) and b)for each V_(H) and V_(L) sequence pair in the repertoire of sequencepairs, and (d) selecting the V_(H) and V_(L) sequence pairs encoding anantibody or antibody fragment which bind to at least one of the vacciniavirus strains or one of the antigens.
 28. A polyclonal cell line capableof expressing a recombinant polyclonal anti-orthopoxvirus antibodyaccording to claim
 1. 29. A polyclonal cell line wherein each individualcell is capable of expressing a single V_(H) and V_(L) coding pair andthe polyclonal cell line as a whole is capable of expressing acollection of V_(H) and V_(L) coding pairs, where each V_(H) and V_(L)coding pair encode an anti-orthopoxvirus antibody.
 30. The polyclonalcell line according to claim 29, wherein said collection of V_(H) andV_(L) coding pairs are generated according to the method comprising: (a)providing a lymphocyte-containing cell fraction from a donor vaccinatedwith an orthopoxvirus or recovering from an orthopoxvirus infection; (b)optionally enriching B cells or plasma cells from said cell fraction;(c) obtaining a population of isolated single cells, comprisingdistributing cells from said cell fraction individually into a pluralityof vessels; (d) amplifying and effecting linkage of the V_(H) and V_(L)coding pairs, in a multiplex overlap extension RT-PCR procedure, using atemplate derived from said isolated single cells; and (e) optionallyperforming a nested PCR of the linked V_(H) and V_(L) coding pairs. 31.The anti-orthopoxvirus recombinant polyclonal antibody according toclaim 1, wherein said anti-orthopoxvirus recombinant polyclonal antibodyis essentially free from immunoglobulin molecules that do not bind toorthopoxvirus antigens.
 32. The anti-orthopoxvirus recombinantpolyclonal antibody according to claim 1, wherein the constant region ofsaid anti-orthopoxvirus recombinant polyclonal antibody belongs to theisotype of IgG, IgA, IgE, IgM, or IgD.
 33. The anti-orthopoxvirusrecombinant polyclonal antibody according to claim 1, wherein theconstant region of said anti-orthopoxvirus recombinant polyclonalantibody belongs to one or two particular immunoglobulin subtypes of IgGor IgA.